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This is to certify that the
dissertation entitled
INTEGRATED DISEASE MANAGEMENT IN TOMATO

PRODUCTION SYSTEMS

presented by
FRANK J . LOUWS

has been accepted towards fulﬁllment
of the requirements for

PLANT PATHOLOGY

 

Ph °D 5 degree in

um FE 14,97 5",”me

Major proiésor

Date Nov tr) MW

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MSU Is An Affirmative Action/Equal Opportunity Institution
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INTEGRATED DISEASE MANAGEMENT IN TOMATO PRODUCTION SYSTEMS
BY

FRANK J. LOUWS

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

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1994

 

ABSTRACT

INTEGRATED DISEASE MANAGEMENT IN TOMATO PRODUCTION
SYSTEMS

BY
FRANK J. LOUWS

Early blight (EB), anthracnose (ANTH), and soil rot (SR), comprise a fungal
disease pathosystem and bacterial—canker, -speck, and -spot, comprise a bacterial
disease pathosystem that limit yield and fruit quality in rnidwest and Ontario tomato
production systems. This thesis developed the premise that disease management
strategies should reﬂect the divergent mechanisms by which the fungi and bacteria
become epidemiological problems. The fungi reside in the agro-ecosystem, incite
disease each year, are the target of regular fungicide applications and ultimately, are
most effectively controlled through farm-level decisions and inputs. Three years of
ﬁeld research demonstrated EB, ANTH, and SR can be effectively managed through
the integration of cultural practices such as conservation tillage and cover crops, that
provided advantages consistent with a sustainable agriculture, and a disease forecasting
model, TOM-CA ST. Select forecast generated chlorothalonil spray schedules did not
compromise yield of marketable fruit but required 45% to 80% fewer sprays compared
to a standard weekly spray program. Reduced-sprays were most effective when
combined with a zone tillage (ZT) system in 1990, a conventional tillage (CT) system
in the absence of rotation in 1991, and ZT or rotation in 1992. _

The bacterial pathosystem is likely to be controlled through genetic resistance

and prevention (i.e. disease free seed and transplants). Effective control is therefore

dependent on knowledge of the genetic diversity of each pathogen. The genetic

 

 

 

 

FRANK J. LOUWS

diversity of a world-wide collection of over 80 isolates of the spot organism,
Xanthomonas campestris pv. vesicatoria (Xcv) was assessed using repetitive DNA
sequences and the polymerase chain reaction (PCR). Based on ﬁngerprint patterns
generated from total genomic DNA, the protocol known as rep-PCR, delineated 4
diverse evolutionary lineages classiﬁed as Xcv. One lineage, designated as Group B,
was newly described as an important component of the spot disease complex in the
midwest.

Integrated disease management in tomato production systems is possible
through the combination of farm level activity, such as conservation tillage and

rotation, and supra—farm-level activity, such as biotechnology based protocols.

Additional Key Words: sustainable agriculture, reduced pesticides, reduced tillage,

REP-, ERIC, BOX-PCR, genetic ﬁngerprinting, bacteria

 

 

 

ACKNOWLEDGEMENTS

Having been surrounded by the love of God, a supportive family and numerous
colleagues and friends, I close this thesis work with gratiﬁcation. I thank my major
professor Christine Stephens for her friendship and for the philosophical framework
that allowed the pursuit of basic and applied research interests. She allowed
considerable freedom that in every way enhanced my education and professional
development. I thank Dr. Frans de Bruijn, Dr. Dennis Fulbright and Dr. Andrew Jarosz
not only for their excellent support and advice as my committee members, but also for
their friendship. I’m also grateful to Dr. Hugh Price and Dr. Jack Kelly for their
expertise and help with the tomato ﬁeld project.

Special thanks to Dr. Mary Hausbeck who went beyond the call of duty in her
new position at MSU. I thank her for her friendship, advise, for the numerous
discussions we had, and for all her help in the preparation of this thesis. Thanks to Dr.
de Zoeten for numerous career oriented discussions and other informal interactions.
My time at MSU was also enriched by numerous other friendships and interactions
with faculty and students. Thanks to my friends at Intervarsity Christian Fellowship,
the Intervarsity Ph.D. Faith and Academic discussion group and the Sustainable
Agriculture discussion group. There was no end to the vigorous and stimulating

discussions.

 

 

 

 

Thanks to Dr. Scott Eisensmith for custom writing statistical software so I
could analyze my data in a timely manner. I also appreciate the ﬁnancial support from
the Natural Science and Research Council of Canada, Dr. Dick Harwood and the CS.

Mott Foundation, the Southwest Research and Extension Station and the HI. Heinz

Co., USA.

I thank my wife and best friend Helen for being so supportive. This thesis
would not have been half the experience without the joy of her love and
companionship. Thanks to my parents and our extended family who have provided so

much support through the last four years.

To God be the glory and I thank him for the open doors and the challenges

provided. All I have comes from him.

”When a farmer plows for
planting, does he plow continually?
Does he keep on breaking
up and harrowing the soil?
When he has leveled the
surface,
does he not sow caraway
and scatter cummin?

Does he not plant wheat in its place,
barley in its plot,
and spelt in its ﬁeld?

His God instructs him
and teaches him the right
way.

All this comes from the
Lord Almighty,
wonderful in counsel and
magniﬁcent in wisdom.”

Isaiah son of Amoz, 725 BC.
Taken from the Holy Bible,

New International Version

c 1973, 1978, 1984 by the
International Bible Society

 

 

 

 

 

 

 

TABLE OF CONTENTS

CHAPTER I

GENERAL INTRODUCTION AND LITERATURE REVIEW

A.INTRODUCTION .................................................................................................... 1
B. LITERATURE REVIEW:INTEGRATED DISEASE MANAGEMENT AND
SUSTAINABLE AGRICULTURE ......................................................................... 3
1. INTEGRATED DISEASE MANAGEMENT ............................................ 3
2. SUSTAINABLE AGRICULTURE ............................................................ 3
3. SELECTED COMPONENTS OF FARM-LEVEL SUSTAINABLE
PRACTICES IN VEGETABLE PRODUCT ION SYSTEMS ..................... 5
a. CONSERVATION TILLAGE .......................................................... 6
b. COVER CROPS ............................................................................... 9
c. REDUCED PESTICIDES ............................................................... 10
C. IIVIPORTANT PATHOSYSTEMS IN THE NORTHCENTRAL TOMATO
PRODUCTION REGION ....................................................................................... 13
l . FUNGAL FOLIAR-FRUIT PATHOSYSTEM ......................................... 13
a. EARLY BLIGHT ............................................................................ 14
i. SYMPTOMS ..................................................................... 15
ii. CAUSAL ORGANISM .............. 16
iii. DISEASE CYCLE AND EPIDEMIOLOGY ................... 17
iv. CONTROL ....................................................................... 18

vi

 

 

 

 

 

 

b. ANTI-IRACNOSE ........................................................................... 20

1. SYMPTOMS ..................................................................... 21

11 CAUSAL ORGANISM .................................................... 21

111 DISEASE CYCLE AND EPIDEMIOLOGY ................... 22

iv. CONTROL ....................................................................... 24
c.RHIZOCTONIA SOIL ROT .......................................................... 25
1. SYMPTOMS ..................................................................... 26

11. CAUSAL ORGANISM .................................................... 26

111. DISEASE CYCLE AND EPIDEMIOLOGY ................... 27

iv. CONTROL ....................................................................... 27

d. FUNGAL FOLIAR-FRUIT PATHOSYSTEMSUMMARY ....... 28
2. THE BACTERIAL PATHOSYSTEM ....................................................... 29

a. BACTERIAL CANKER, BACTERIAL SPOT AND

BACTERIAL SPECK ..................................................................... 29
i. SYMPTOMS ..................................................................... 29
ii. CAUSAL ORGANISM .................................................... 30
iii. DISEASE CYCLE AND EPIDEMIOLOGY ................... 32
iv. CONTROL ....................................................................... 33
b. SUMMARY OF BACTERIAL PATHOSYSTEM ....................... 35
D. ASSESSING POPULATION GENOTYPIC DIVERSITY OF PLANT
PATHOGENS ......................................................................................................... 35
LITERATURE CITED .............................................................................................. 40
vii

 

 

 

CHAPTER II

INTEGRATED MANAGEMENT OF EARLY BLIGHT, ANTHRACNOSE
AND SOIL ROT OF TOMATO WITH REDUCED FUNGICIDE USAGE
AND CULTURAL PRACTICES

ABSTRACT ............................................................................................................... 57

INTRODUCTION ...................................................................................................... 58

MATERIALS AND METHODS .............................................................................. 60

RESULTS .................................................................................................................. 71

DISCUSSION .......................................................................................................... 107

LITERATURE CITED ............................................................................................ 115
CHAPTER III

CONSERVATION TH1LAGE AND ROTATION TO CUCUMBER IN
TOMATO PRODUCTION SYSTEMS

ABSTRACT ............................................................................................................. 120

INTRODUCTION ............................................................. . ...................................... 121

MATERIALS AND METHODS ............................................................................ 122

RESULTS ................................................................................................................ 126

DISCUSSION .......................................................................................................... 144

LITERATURE CITED ............................................................................................ 148
CHAPTER rv

MAJOR DISTINCTIONS IN GENOMIC STRUCTURE DETECTED BY
REP-PCR FINGERPRINTING SEPARATE STRAINS CLASSIFIED AS
XANTHOMONAS CAMPESTRIS PV. VESICATORIA INTO AT LEAST

FOUR GROUPS.
ABSTRACT ............................................................................................................. 151
INTRODUCTION .................................................................................................... 152

viii

 

 

 

 

 

 

 

MATERIALS AND METHODS ............................................................................ 154

RESULTS ................................................................................................................ 159

DISCUSSION .......................................................................................................... 181

LITERATURE CITED ............................................................................................ 188
CHAPTER V

CONCLUSIONS AND FUTURE RESEARCH

CONCLUSIONS ....................................................................................................... 192
FUTURE RESEARCH ...................... ‘ ...................................................................... 195
LITERATURE CITED ............................................................................................ 198
APPENDIX A: .................................................................................................................... 200
APPENDIX B: .................................................................................................................... 204
APPENDIX C: .................................................................................................................... 208
APPENDIX D: .................................................................................................................... 210

ix

 

 

 

LIST OF TABLES

CHAPTER H

TABLE 1: Number of hours of leaf wetness at a given temperature range required for
each disease severity value (DSV) ................................................................................... 65

TABLE 2: Date of harvest, date and rate of ethrel treatment, fungicide treatment,
number of fungicide applications, and date of initial fungicide application in years
1990 to 1992 ...................................................................................................................... 66

TABLE 3: Mean temperature and rainfall for Southwest Michigan Research and
Extension Center for 1990, 1991 and 1992 ..................................................................... 71

TABLE 4: Mean squares from analysis of variance for log area under disease progress
curve (AUDPC) for foliar incidence of early blight in processing tomato (PRT) or
fresh market tomato (FMT) in 1990. .............................................................................. 73

TABLE 5: Backtransformed 1990 values for area under the disease progress curve for
processing tomato and fresh market tomato. Analysis of variance and mean separation
was based on log transformed data. ................................................................................ 78

TABLE 6: Mean squares from analysis of variance for log area under disease progress
curve (AUDPC) for foliar incidence of early blight in processing tomato (PRT) or
fresh market tomato (FMT) in 1991 ................................................................................ 81

TABLE 7: Backtransformed 1991 values for area under the disease progress curve for
processing tomato and fresh market tomato. Analysis of variance and mean separation

was based on log transformed data .................................................................................. 82

TABLE 8. Mean squares from analysis of variance for log area under disease progress
curve (AUDPC) for foliar incidence of early blight in processing tomato (PRT) or
fresh market tomato (FMT) in 1992 ............................................................................... 84

TABLE 9: Backtransformed 1992 values for area under the disease progress curve for
processing tomato and fresh market tomato. Analysis of variance and mean separation
was based on log transformed data. . ............................................................................ 85

 

 

 

TABLE 10. Percent plants with early season incidence Q SE) of a leaf spot and
Alternaria solani collar rot on processing and fresh market tomato plants,1991 .......... 88

TABLE 11A: Proportion of total percent fruit with mold due to early blight,
anthracnose, and soil rot observed for processing tomato in 1990 to 1992 ................... 90

TABLE 11B: Proportion of total percent fruit with mold due to early blight,
anthracnose, and soil rot observed for fresh market tomato in 1990 to 1992 ................ 90

TABLE 12: Mean squares from analysis of variance for percent fruit with mold due to
early blight, anthracnose or soil rot in processing tomato (PRT) or fresh market tomato

(FMT) in 1990 ................................................................................................................... 92

TABLE 13: Mean squares from analysis of variance for percent fruit with mold due to
early blight, anthracnose or soil rot in processing tomato (PRT) or fresh market tomato

(FMT) in 1991 ................................................................................................................... 93

TABLE 14. Post—harvest incidence (%) of fully red NO.1 fresh market tomato fruit
with symptoms of anthracnose in 1991 ............................................................................ 98

TABLE 15: Mean squares from analysis of variance for percent fruit with mold due to
early blight, anthracnose or soil rot in processing tomato (PRT) or fresh market tomato
(FMT) in 1992 ................................................................................................................. 100

TABLE 16: Pearson’s correlation coefﬁcient (r), intercept, Slope, standard error (SE) of
the slope and signiﬁcance value of the correlation of the relationship between
incidence of fruit mold due to early blight and anthracnose in processing (PRT) and
fresh market tomato (FMT) in 1990, 1991, and 1992 ................................................... 106

CHAPTER III

TABLE 1: Date of harvest, date and rate Of ethrel treatment, fungicide treatment,
number of fungicide applications, and date of initial fungicide application in years
1990 to 1992 .................................................................................................................... 124

TABLE 2: Percent surface rye residue (j; standard error) in plots conventionally tilled
(CT) or managed by a zone tillage (ZT) system in 1992 ............................................. 126

TABLE 3: Mean squares from analysis of variance for yield and fruit quality of fresh
market tomato (FMT) in 1990 ........................................................................................ 128

TABLE 4A. Effect of tillage on marketable yield (metric tonnes) of fresh market
tomato in 1990 ................................................................................................................. 128

 

 

 

TABLE 48. Effect of fungicide treatment on marketable yield (metric tonnes) Of fresh
market tomato in 1990. .................................................................................................. 129

TABLE 4C. Means of cull weight of fungicide x tillage interaction of fresh market

tomato in 1990 ................................................................................................................. 129
TABLE 5: Mean squares from analysis of variance for yield and fruit quality of
processing tomato (PRT) in 1990 ........................................................................................ 135
TABLE 6: Effect of tillage on marketable yield of processing tomato cv. OHIO 7870 in
1990 ....................................................................................................................................... 135
TABLE 7: Mean squares from analysis of variance for yield and fruit quality of fresh
market tomato (FMT) in 1991 ............................................................................................. 137
TABLE 8A. Effect of tillage on marketable yield of fresh market tomato in 1991 ......... 138
TABLE SB. Effect of fungicide treatment on marketable yield of fresh market tomato in
1991 ....................................................................................................................................... 138
TABLE 9: Mean squares from analysis Of variance for yield and fruit quality of
processing tomato (PRT) in 1991 ........................................................................................ 139
TABLE 10: Effect of tillage on marketable yield of processing tomato cv. Heinz 8780 in
1991 ....................................................................................................................................... 139
TABLE 11: Effect of tillage on yield of pickling cucumber cv. Flurry in 1991 .............. 140
TABLE 12: Mean squares from analysis of variance for yield and fruit quality of fresh
market tomato (FMT) in 1992 ............................................................................................. 142
TABLE 13A: Effect of rotation and tillage on marketable fruit of fresh market tomato in
1992 ....................................................................................................................................... 143
TABLE 138. Effect of fungicide treatment on marketable yield of fresh market tomato
in 1992 ................................................................................................................................... 143
TABLE 13C: Means of cull fruit Of selected fungicide x rotation treatment
interactions ............................................................................................................................ 143
CHAPTER IV

TABLE 1: Bacterial isolates or DNA used in this study and associated information ...... 155

xii

 

 

 

APPENDIX A

TABLE A1: Mean squares from analysis of variance for yield and fruit quality of fresh
market tomato (FMT) in 1992 using 4 replications ............................................................ 200

TABLE A2: Effect of rotation and tillage on marketable fruit of fresh market tomato in
1992 using 4 replications ...................................................................................................... 201

TABLE A3. Effect of fungicide treatment on marketable yield of fresh market tomato in
1992 using 4 replications ...................................................................................................... 201

TABLE A4: Mean squares from analysis of variance for yield and fruit quality of
processing market tomato (PRT) in 1992 using 4 replications .......................................... 202

TABLE A5: Effect of tillage on marketable yield of processing tomato cv. Heinz 8780
in 1992 using 4 replications ................................................................................................. 203

TABLE A6. Effect of fungicide treatment on marketable yield of processing tomato in
1992 using 4 replications ...................................................................................................... 203

xiii

 

 

 

LIST OF FIGURES

CHAPTER H

FIGURE 1: Summary of the overall 3 year crop production system employing zone
tillage (ZT) and rotation of tomato with cucumber. Solid lines represent crop growth.
Dotted lines represent windows of preferred time periods for agronomic inputs. Fields
are conventionally tilled (CT) commencing year one and not subject to CT again until
after the cucumber harvest in year two. After a mustard crop, CT is used once more
and the ﬁeld is planted back to tomato. Intensive two year rotations are common with
some growers in SW Michigan ........................................................................................ 64

FIGURE 2: Disease progress curves of percent defoliation of processing tomato plants
estimated weekly in 1990, 1991, and 1992. The insert graph of the 1990 and 1991
ﬁgures represent disease data of zone tillage (ZT) and conventional tillage (CT) plots
transformed with the logistic model. Values for signiﬁcance of intercept and slope are
given in the text. R- = no rotation; R+ = with rotation .................................................. 75

FIGURE 3: Disease progress curves of percent defoliation of fresh market tomato
plants estimated weekly in 1990, 1991, and 1992. The insert graph of the 1990 and
1991 ﬁgures represent disease data of zone tillage (ZT) and conventional tillage (CT)
plots transformed with the logistic model. Values for signiﬁcance of intercept and
slope are given in the text. - = no rotation; R+ = with rotation .................................. 77

FIGURE 4:Incidence and severity of early blight symptoms recorded on processing
tomato plants on July 16, 1992 ........................................................................................ 89

FIGURE 5: Incidence of fruit mold expressed as percent of total weight of processing
tomato fruit harvested in 1990 and 1991. Bars represent the main effect of each
treatment (i.e. mean of CT and ZT plots combined). Bars with the same letter are not
signiﬁcantly different based on protected LSD value. P value indicates the level of
signiﬁcance between the main effect of CT as compared to ZT on incidence of fruit
mold ................................................................................................................................... 95

FIGURE 6: Incidence of fruit mold expressed as percent Of total weight of fresh
market fruit harvested in 1990 and 1991. Bars represent the main effect of each
treatment (i.e. mean of CT and ZT plots combined). Bars with the same letter are not
signiﬁcantly different based on protected LSD value. P value indicates the level of
Signiﬁcance between the main effect of CT as compared to ZT on incidence of fruit

xiv

 

 

 

 

 

mold ................................................................................................................................... 9 7

FIGURE 7: Incidence of fruit mold expressed as percent of total weight of processing
(top) and fresh market (bottom) tomato fruit harvested in 1992. The 3 way interaction
between rotaton, tillage and fungicide treatment was signiﬁcant. Each point is the
mean of 2 replications in conventional tillage (CT) or zone tillage (ZT) plots combined
with a one year rotation or not rotated .......................................................................... 102

FIGURE 8. Relationship of incidence of fruit mold, due to early blight (top) or
anthracnose (bottom), to non-transformed Area Under the Disease Progress Curve
(AUDPC) values in processing tomato (left) and fresh market tomato (right).
Equations provide the estimated intercept, slope, error mean sauare (EMS) and
coefﬁcient of determination (R2) for linear regression analysis between early blight or
anthracnose and AUDPC values ..................................................................................... 105

CHAPTER HI

FIGURE 1: Effect of tillage and rotation on fresh market cv. ’Pik—Rite’ tomato yields
harvested each week ........................................................................................................ 131

FIGURE 2: Effect of fungicide on fresh market cv. ’Pik-Rite’ tomato yields harvested
each week ........................................................................................................................ 133

CHAPTER IV

FIGURE 1. Agarose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria using primers
corresponding to REP (lanes 1 to 6), BOX (lanes 7 to 12) and ERIC (lanes 13 to 18)
sequences. Six ul of PCR products were loaded in each lane. A typical Group A
pattern (lanes 1,7 and 13), Group B pattern (lanes 2,8 and 14), the Group C pattern
(lanes 3,9 and 15) and Group D pattern (lanes 4&5, 10&11 and 16&17) are displayed.
The Iight— and left-most lane contain DNA size markers (1 kb ladder, Gibco-BRL)
indicated in base pairs. Arrowheads identify Similarities or differences among selected
isolates as outlined: in the text. PCR bands were resolved on 1.5% agarose gels stained
with ethidium bromide .................................................................................................... 162

FIGURE 2. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria Group A (Top) and
Group B (Bottom), using primers corresponding to BOX sequences. Other details are
as outlined in the legend of Figure 1 ............................................................................. 164

FIGURE 3. A garose gel electrophoresis of ﬁngerprint patterns Obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria Group A using primers
corresponding to ERIC sequences. Other details are as outlined in the legend of Figure
1 ........................................................................................................................................ 168

 

 

 

 

 

 

 

 

FIGURE 4. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria Group B using ERIC
(A) and REP (B) primers. Other details are as outlined in the legend of Figure l ..170

FIGURE 5: Agarose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria Group A using primers
corresponding to REP sequences. Other details are as outlined in the legend of Figure
1 ........................................................................................................................................ 172

FIGURE 6. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates classiﬁed as Xanthomonas campestris pv. vesicatoria Group D
(lanes 1&2) as compared to patterns generated from representative Group A isolates
(lane 5) and isolates of Xanthomonas campestris pv. campestris (lanes 3&4) using

BOX primers. Other details are as outlined in the legend of Figure 1 ....................... 175
FIGURE 7: Increase (mm) in the radius of cleared zones indicating starch hydrolysis
by selected isolates of Xanthomonas campestris pv. vesicatoria .................................. 180
APPENDIX B

FIGURE B1: Effect of tillage and fungicide on the incidence of bacterial speck ....... 205

FIGURE B2: Effect of tillage on the early infestation of plots by the Colorado potato

bettle ................................................................................................................................ 207
APPENDIX C
Direct analysis of bacteria on media and in plant lesions ............................................. 209

 

 

 

 

 

 

 

CHAPTER I

GENERAL INTRODUCTION AND LITERATURE REVIEW

A. INTRODUCTION

Over 25,000 ha Of tomato are grown in Michigan, Ohio, Indiana and Ontario
(the northcentral production region) with an estimated farm gate value of 145 M US
dollars (OMAF 1990; USDA 1990). Current yields of up to 70 T ha’1 are not
uncommon in the processing tomato industry, as compared to 5 to 8 T ha’l recorded
by Brown in 1929 (Brown 1929). Crop productivity has been enhanced through
superior tomato cultivars, specialization and mechanization, intensive tillage of soils
combined with high fertilizer inputs, and signiﬁcant chemical-dependent advances in
weed, insect and disease control. However, the industry currently faces numerous
challenges. Synthetic chemicals are facing an unprecedented challenge including
consumer, regulatory agency, environmental and grower safety concerns. Likewise,
intensive tillage of the land and energy-intensive inputs are seen as counter-productive
to long-term sustainability. A movement toward a sustainable agriculture impinges on
current chemical dependent disease control and agronomic practices even though there
is a lack of adequate alternatives such as cultural, genetic or biological disease control
strategies and reduced tillage systems.

Fungal and bacterial diseases limit yield and product quality yearly and are
favored by the climate of the northcentral region. This thesis focuses on integrated

disease management of ﬁeld-tomato diseases within a context of current tomato

 

 

 

77

 

 

 

 

2

production systems. Field research was conducted in southwest Michigan where many
growers farm on light sandy. soils and follow an intense biennial cropping sequence of
tomato and cucumber crops. With regard to disease management, this thesis takes two
approaches. The ﬁrst approach may be considered ” grower dependent” integrated
management. Management of diseases within this context occurs primarily at the
individual farm enterprise level. Each grower manages knowledge and material inputs
for disease control within their Site speciﬁc production system. The second approach
may be considered ”industry dependent" integrated management where ”industry”
refers to the tomato industry as a whole including private and public institutions. At
this level, inputs are ﬁrst of all knowledge based and are integrated for disease
management before the seed anives at the farm gate.

The objective of this study was to: 1) minimize the number of fungicide
applications required for control of early blight, anthracnose and soil rot of tomato
through the use of tillage, a green manure crop, and weather timed frmgicide sprays
within a biennial (tomato/cucumber) conservation tillage production system, without
compromising fruit quality and yield, and,

2) to assess the genetic diversity of bacterial pathogens of tomato, using the
bacterial spot organism as a model, and ascertain how genetic diversity impacts
industry dependent disease management strategies.

The literature review will highlight components included in the tomato

production system within a broader context of sustainable agriculture.

 

 

 

 

 

3

B. LITERATURE REVIEW:INTEGRATED DISEASE MANAGEMENT AND

SUSTAINABLE AGRICULTURE

1. INTEGRATED DISEASE MANAGEMENT

Integrated disease management is a form of the generic integrated pest
management (IPM) or integrated pest control (IPC) and is deﬁned as ”a pest
management system that, in the context of the associated environment and the
population dynamics of the pest species, utilizes all suitable techniques and methods in
as compatible manner as possible and maintains the pest populations at levels below
those causing economic injury” (FAO/UNEP 1984). The concept of IPM has
progressively evolved since Stem et al. (1959) ﬁrst introduced it. Originally, IPM grew
in response to increasing crop damage and pest resistance to chemical based controls
(Cooley 1993). Today, the concept has evolved to include broad goals including
environmental and social issues, and has become a mainstay in the sustainable

agriculture movement (Poincelot 1986).

2. SUSTAINABLE AGRICULTURE

Sustainable agriculture is a philosophy of agriculture reflectin g human goals
and embraces a broad range of definitions found throughout the literature. Most
deﬁnitions of sustainability encompass rural community and farm family socio-
economic aspects, preservation of non-renewable agrO-ecosystem resources (water, soil
and biodiversity), the need for an adequate food and ﬁber supply to meet the needs of
a growing population, and optimization of long term farm enterprise proﬁtability and

productivity, presumably over an indeﬁnite time frame. Allen et al. (1991) perceived

 

 

 

 

 

4

that sustainable agriculture has a focus on the entire global food and agriculture
system that does not just include environmental and economic viability, but also social
justice for all sectors of society.

The 1990 United States farm bill has less of a global perspective and deﬁned
sustainable agriculture as ”an integrated system of plant and animal production
practices having site speciﬁc application that will, over the long term:satisfy human
food and ﬁber needs; enhance environmental quality and the natural resource base
upon which the agriculture economy depends; make the most efﬁcient use of non-
renewable resources and integrate, where appropriate, natural biological cycles and
controls; sustain the economic vitality of farm operations; and enhance the quality of
life for farmers and society as a whole” (Bird 1993). Deﬁnitions lose a sociological
tone and progressively become more practical oriented when applied to the actual
process of farming. For example, Francis et al. (1987) deﬁne sustainable agriculture as
a ”result of a management system which helps the producer to choose the hybrids
and/or varieties, soil fertility packages including rotations, pest management
approaches, and cropping sequences to reduce costs of purchased inputs, minimizing
the impact of the system on the immediate off-farm environment, and provide for a
sustained level of production and proﬁt from farming”.

At the farm enterprise and ﬁeld level, Fretz et a1. (1993) suggest sustainable
agriculture requires more of a ”process oriented ” and ”problem—solving" mentality as
compared to a ”product oriente " mentality. Information and management are said to
substitute for non-renewable, energy-intensive inputs. At the farm level, many

published works envision improved crop rotations, use of legumes in cover crop

sequences, improved nutrient cycling, and a livestock component. Wien (1990) notes

 

 

 

 

 

 

5

that farm-level sustainability is not stepping back into the past but requires ”a much
more sophisticated and detailed understanding of the agriculture ecology
...[including]...pest and predator populations and their dynamics...”. The latter
deﬁnition of Francis et al. and expanded on by Fretz et a1. and Wien provided a

functional framework for this thesis.

3. SELECTED COMPONENTS OF FARM-LEVEL SUSTAINABLE
PRACTICES IN VEGETABLE PRODUCTION SYSTEMS

Sustainable farming practices have not been adopted with equal success in
vegetable production systems as compared to ﬁeld crop or mixed farming Operations
(Kelly 1990). For example, systems that include tomato production, especially fresh
market tomato production systems, are specialized and do not have crop diversity or
land resources to include long crop rotations, legume based crops or reduced tillage
practices. Considerable research is required to make vegetable production systems
more sustainable and less input intense. For more than a decade, researchers at
Michigan State University have examined the potential of a more sustainable tomato
production system (Barnes and Putnam 1983; Drost 1983; Grajauskis 1990; Jardine et
a1. 1988; Price and Baughn 1987; H. Price and C.T. Stephens unpublished; Putnam
1990). Key components include conservation tillage practices, use of cover and green
manure crops, and reduced pesticide input.

Conservation tillage systems and use of cover crops are integral components of
sustainable production. The practices have many advantages but also some draw backs
(Coolrnan and Hoyt 1993; Kenimer et a1. 1986; Morse 1993; Phillips et a1. 1980;

Sarrantonio 1992; Sherman 1992; Spieser 1983). Advantages include dramatic

 

 

 

 

 

 

6

reductions in wind and water soil erosion; enhanced water use efﬁciency; improved
soil moisture content; enhanced soil productivity; decreased dependence on non—
renewable energy; increased nutrient recycling; elevated microbial activity; decreased
pesticide losses and improved timing of harvesting and planting. Disadvantages include
increased soil compaction problems; altered weed and disease populations that may
require additional pesticide inputs; reduced germination or crop stands, and decreased
soil temperatures. Success with speciﬁc tillage practices and cover crops is inﬂuenced
by soil type, cropping system, environment and choice of cover crop among other

components (Benoit and Lindstrom 1987; Sarrantonio 1992).

a. CONSERVATION TILLAGE

Conservation tillage is defined as ”any tillage and planting system that retains
at least 30% residue cover on the soil surface after planting (I-Iiemstra and Bauder
1984). Variations, as deﬁned by the Soil Science Society Of America (1978), include:
no-tillage (NT) — "a crOp production system whereby a crop is planted directly into a
seedbed not tilled since harvest of the previous crop"; and minimum tillage - ”the
minimmn soil manipulation necessary for crop production or meeting tillage
requirements under the existing soil and climatic conditions”. Conventional tillage
(CT) reﬂects the normal procedures for crop production in a given region but usually
consists of a primary deep tillage operation (moldboard plow) followed by one or
more passes of secondary tillage (Soil Science Society of America 1978; Phillips et a1.
1980).

Conservation tillage practices are common and successful in many ﬁeld crop

production systems (Phillips et a1. 1980) but provide variable results in tomato

 

 

 

 

 

 

7

production systems. Beste (1976) observed yields of tomatoes direct seeded into a rye
stubble mulch (NT) were equal to yields in a CT system. Knavel et al. (1977)
transplanted tomatoes for two years into a sod cover and a third year into a wheat
mulch. The sod and wheat NT systems did not affect transplant survival but decreased
and had no affect on yield, respectively, as compared to Cl‘. Shelby et al. (1988)
transplanted tomatoes into a desiccated wheat cover and obtained nearly twice the
marketable yields as compared to CT. In a second year, NT and CT yields were
similar.

Doss et al. (1981) observed marketable yield of fresh market staked tomatoes
tended to decrease as the amount of tillage decreased. Treatments included in row ‘
chiseling combined with direct ﬁeld setting of tomato transplants into a rye mulch
(NT), strips of incorporated rye mulch, or CT plots. They noted early plant growth
decreased in rye plots. The impact of NT using a rye cover crop was also noted by
Price and Baughn (1987) and Grajauskis (1990). Early plant growth and fresh market
tomato yields were consistently depressed in their NT system.

Associated yield decline with rye mulch cover has led to modiﬁed minimum
tillage systems such as strip tillage (ST). McKeown et a1. (1988) tilled strips into
killed rye or cat cover crops approximately 3 weeks prior to transplanting tomatoes.
Yields in all plots were similar for 2 years but depressed in rye ST plots a third year.
Grajauskis (1990) employed a modiﬁed ST system. Rye strips were incorporated in
early spring and the remainder of the rye was desiccated at a later date prior to
planting. Soil was fractured directly below where tomato plants were to be established
(zone tillage ZT). The combination of ST and ZT enhanced marketable yields as

compared to NT or CT and enhanced plant productivity was associated with a more

 

 

 

 

8

extensive root system in ZT plots (Grajauskis 1990). Hedgewood et al. (1978) also
noted subsoiling beneﬁted tomato productivity. Sumner et al. (1981) have associated
subsoilin g with enhanced root penetration and greater efﬁciency of nutrient uptake as
opposed to impact on root disease incidence.

Weed control is a recurring problem with reduced tillage in vegetable
production systems (Knavel et al. 1977; Putnam 1990). In transplant tomato
production, pre-plant incorporation of triﬂuralin provides early season control of

germinating weed seeds but triﬂuralin cannot be applied in reduced tillage systems.

 

However, recent advances in post emergent selective herbicides allow for adequate
weed control in reduced tillage systems (Putnam 1990; Wallace and Bellinder 1992).

Shelby et a1. (1988) demonstrated the potential of metribuzin and sethoxydirn for post

 

emergent control of broad leaf weeds and grasses, respectively, in tomatoes.

Most conservation tillage experiments in vegetable production systems do not
include a plant pathology component (Sumner et al. 1986). Jardine et al. (1988)
observed a higher incidence of bacterial speck on tomato plants in NT compared to
CT plots infested with infected debris the previous fall. McKeown et al. (1988)
observed bacterial speck incidence was higher in ST in 1 out of 3 years and plant

parasitic nematode populations were stimulated by ST. Sumner et al. (1981 & 1986)

 

have observed that conservation tillage practices increase, decrease or have no effect
on plant diseases. They summarize that tillage irnpacts Rhizoctonia populations but
generally not Fusarium, Pythium or nematode populations. In general, they observed
root diseases are affected more by the previous crop than by tillage practices. Few
studies have examined the impact tillage has on the incidence of foliar diseases of

vegetables. Surrmer et al. (1986) surmise that burial of crop debris may reduce initial

_—4

 

 

 

 

 

 

9

inoculum but have no effect on secondary spread of foliar pathogens, except indirectly
through altered host productivity and reaction to infection.
b. COVER CROPS

Cover crops have been used in crop production systems for thousands of years
to enhance soil fertility and physical properties (Sarratonio 1992). Numerous cover
crops have proven successful in vegetable production systems (Sherman 1992) and
choice of cover crop is dependent on the growers goals. Le gume crops provide

nitrogen and are preferred for long-term sustainability (Frye and Blevins 1989).

 

However, legume crops require relatively long growth periods to achieve beneﬁts. This

often precludes their use in vegetable production systems. Rapid growing cover crops

 

have the advantage of quickly tying up soil nutrients and are most suitable to ﬁll
windows of opportunity in vegetable production systems (Sarrantonio 1992). Rye
(Secale cereale L.) is often preferred. Fall sown rye is winter hardy and rapidly
acquires biomass in the early spring as compared to oats and wheat.

Rye mulch can act as a smother crop to reduce weed populations. Rye residue
also releases allelopathic substances that can be exploited for early-season weed
control (Barnes and Putnam 1987; Putnam 1990) although allelopathy may also be
responsible for decreased tomato productivity (Grajauskis 1990). Putnam (1990)
summarizes that rye on the soil surface releases chemicals that are highly inhibitory to
dicotyledonous weed seedlings and offers variable control of grassy weeds. Weed
management systems have been effective in tomatoes using rye residue for early
season control and ”rescue" treatments of metribuzin or sethoxydirn applied aS-needed
for control of later-season weeds (Putnam 1990; Wallace and Bellinder 1992). Putnam

(1990) notes reduced rates of post- emergent herbicides can be used with optimum

___—#l

 

 

 

 

10

timing. Such a system requires close monitoring of weed populations.

Cover crops have also been exploited to reduce insect damage (Bugg 1992) and
wind damage to plants (Spieser 1983; Beste 1973). High winds are associated with
decreased yield potential in tomatoes (Arrnburst et al. 1969) and sand blasting injury
which has been associated with increased incidence of disease in tomato ﬁelds (V akili
1967; Rotem 1965).

Limited research has been published concerning the impact of traditional cover
crops on disease incidence in vegetable cropping systems. A rye cover crop reduced
early-season severity of corky root rot of lettuce but this may have been associated
with altered soil physical properties (van Bruggen et al. 1990). In contrast, plant
members of the Cruciferae family have proven activity against plant pathogens
surviving in soils (Mojtahedi et al. 1993; Muehlchen et al. 1990). Sulphur containing
glucosinolates present in tissues of Cruciferae plants hydrolyze enzymatically to form
a number of volatile compounds including isothiocyanate, chemical structures similar
to those used in commercial fumigants. The sulphur containing compounds are
speculated to diffuse in soil and act as fumigants against soilbome pathogens (Lewis

and Papavizas 1970).

c. REDUCED PESTICIDES

Public concern, farm proﬁtability, cancellation of registered fungicides and lack
of new chemistry, restrictive legislation, and pest resistance (Merwin and Pritz 1993;
Stephens 1990) is forcing vegetable growers to reduce pesticide inputs.

Currently, fungicides are applied on nearly 100% of tomato hectarage in the

northcentral production region (Precheur et al. 1992). Most growers follow the

 

 

 

 

 

 

 

11

standard recommendation to initiate sprays when fruit ﬁrst set and to apply subsequent
sprays every 7 to 14 days even if the risk of disease is zero. Up to 12 or more
applications are required each season. However, data over the last several years
suggest a reduced number of precise timed fungicide applications can be used to
control fruit rots in tomato, an approach more compatible with goals of a sustainable
agriculture.

Disease forecasting systems are designed to determine the need for the
initiation and/or timing of subsequent pesticide applications for the purpose of
reducing disease incidence and efﬁcient use of resources. Early blight of tomato is a
”classic” example of research efforts to design efﬁcient spray programs. Martin (1920)
noted early season sprays (in this case copper based ”fungicides”) could be eliminated
without compromising disease control. Horsfall and Heuberger (1942) likewise
concluded, for the northeastern production region, ”July 10 is early enough [to initiate
a spray program] in most years”. During the late 1970’ , control was evaluated using
the ”Massive Dosage Technique” (Pitblado 1992; Stevenson 1977). The primary goal
of massive dosage, prior to pesticide issues moving to the forefront of the public
conscience, was to reduce time devoted to fungicide applications. Fungicides were
applied at 2 to 3 fold rates on a reduced schedule, compared to recommended rates
every 7 to 10 days. Limited success was achieved and multiple applications continued
to prove more successful (Stevenson 1977).

Waggoner and Horsfall (1969), in their classic publication "EPIDEM:’, designed
a Fortran based computer simulation of early blight disease progress. The program was
based on considerable original work, previously published work and over a decade of

notes on monitored early blight epidemics. EPIDEM, and work by others, was

 

 

 

 

 

12

synthesized to formulate FAST, a Forecast system for Alternaria Solam' on Tomato
(Madden et al. 1978). FAST incorporated two empirical models utilizing daily
maximum and minimum air temperature, hours of leaf wetness, maximum and
minimum temperature during the wetness period, and hours of relative humidity
greater than 90% and rainfall. FAST effectively controlled early blight with fewer
spray applications as compared to weekly applications (Madden et al. 1978;
Pennypacker et al. 1983).

The multiple environmental parameters required by FAST and the cumbersome
equipment (Taylor dew meter and hygrotherrnograph) required to monitor the weather
patterns, limited the application and general use of FAST. From 1983 to 1988, Dr.
Ron Pitblado at the Ridgetown College of Agricultural Technology, Ridgetown,
Ontario, evaluated and modiﬁed FA ST to be less complex and, effective for control of
early blight, anthracnose and Septoria leaf spot (Pitblado 1988; 1992). Pitblado Simply
used a table, devised by Madden et al. ( 197 8), to calculate daily disease severity
values (DSVS) based on the average temperature during hours when foliage are wet.
The modiﬁed program was called Tom-Cast for TOMato disease foreCA STer.
According to the Tom-Cast model, the first Spray is applied on July 11 or earlier if 28
days have lapsed since transplanting and if the DSV has a cumulative value of 35 for
tomatoes planted before 23 May and 45 for tomatoes planted after 23 May.

Subsequent sprays are applied after the accumulation of a predetermined DSV
threshold since the last fungicide application. The last fungicide application Should be
made 14 days prior to harvest in ﬁelds with no recent history (last 2 years) of
tomatoes and with a low incidence of disease or, 10 days in ﬁelds following minimal

or no crop rotation (Pitblado 1992).

 

 

 

13

Simple data loggers, such as the Omnidata DP~223 (Omnidata Co. Logan UT)
that measure leaf wetness and hourly average temperature, the parameters used to
calculate DSVs, can be deployed in tomato ﬁelds to provide regional or local spray
application recommendations. This enhances the potential of Tom-Cast to be regionally

deployed for the control of fungal pathogens.

C. IMPORTANT PATHOSYSTEMS IN THE NORTHCENTRAL TOMATO

PRODUCTION REGION

A pathosystem is deﬁned as a component of a crop production system
comprised of a host and any sub-set of pathogen(s) (Robinson 1976). Speciﬁc tomato
pathogens encountered in the northcentral production region can be grouped into
pathosystems based on their biology, epidemiology and disease management strategies.
One pathosystem is comprised of Verticillium and F usarium wilt, soilbome fungi that
are effectively controlled through genetic resistance deployed in commercial cultivars.

Two other important pathosystems and studied in this thesis include the fungal foliar-

 

fruit pathosystem and the bacterial pathosystem.

l. FUNGAL FOLIAR-FRUIT PATHOSYSTEM
Economically important diseases that affect the foliage and fruit of tomato

include early blight, anthracnose and Rhizoctonia soil rot.

 

 

 

 

14

a. EARLY BLIGHT
The early blight (EB) pathogen was first isolated in New Jersey in 1882 from
dying potato (Solanum tuberosum L.) leaves (Ellis and Martin 1882). In 1892 the same
fungus was shown to be a pathogen of tomato. Several binomials occur in the
literature but the most common is Alternaria solam' with the authorities (Ellis &
Martin) Sorauer or, (Ellis & Martin) Jones & Grout. The literature pertaining to A.
solam’ is extensive and has recently been reviewed by Pscheidt and Stevenson (1986).
Early blight is widespread in tropical and temperate zones (Ellis & Gibson
1975) and occurs wherever tomatoes are grown (Rands 1917; Jones et al. 1991). Early
blight is particularly destructive in temperate humid climates such as the northcentral
region and semi-arid climates where nightly dew is frequent and moisture requirements
favor disease development (Sherf & MacNab 1986; Rotem & Reichert 1964).
Premature defoliation of tomato leaves is the primary effect of EB. Resultant
impact on fruit yield and quality varies with environmental conditions, cultivar grown,
geographic location, amount and time of arrival of inoculum, and defoliation severity
(Basu 1974; Brammall 1993; Horsfall & Heuberger 1942; O'Leary 1985; Shoemaker
1976; 1980). Losses in marketable yield of fresh market tomato in North Carolina and
in the absence of fungicide applications can be as high as 70% (Shoemaker 1976;
1980). In contrast, in Ontario during the 1991 and 1992 growing seasons, marketable
yield Of 13 fresh market tomato cultivars was not affected by EB epidemics even in
the absence of fungicide sprays (Brammall 1993). Tomato plants can tolerate
signiﬁcant levels of defoliation before yields are detrimentally affected (Basu 1974;

Ferrandino & Elmer 1992).

 

Yields of processing tomato in the rnidwest have been reduced up to 35% in

 

 

 

15

the absence of control (Sherf & MacNab 1986). Aesthetic appearance is not critical
with processing as compared with fresh market tomatoes, but high mold counts
depress the acceptibility and price of tomatoes at the processing plant (Precheur et al.

1992).

i. SYMPTOMS

Altemaria solani attacks all above ground tissue (Sherf & MacNab 1986) and
has recently been reported to incite a root rot (Patterson 1991). Above ground
symptoms have been described as collar rot and early blight.

Collar rot is not common in the northcentral production region but is more
commonly associated with the southern production of tomato seedlings in open ﬁelds
(Moore 1942; Pritchard and Porte 1921). Seedlings develop dark, sunken stem lesions
close to the soil line and lesions can expand to girdle and kill plants or decrease
productivity (Jones et al. 1991). Leaf spot symptoms are diagnostic for early blight.
Symptoms are ﬁrst observed on the lower foliage but progress to the upper foliage as
the plant matures. Complete defoliation ensues in unchecked epidemics. Lesions ﬁrst
appear as small brownish black spots that expand up to 2 cm or more in diameter or
become angular when restricted by leaf veins. Spots develop a series of concentric
dark rings that give a characteristic "target spot" appearance. Spots are often
surrounded by chlorotic tissue associated with phytotoxin production. As spots enlarge
and coalesce infected leaves wither and abscise. Extensive defoliation can result in
sunscald injury to fruit (Sherf & MacNab 1986).

Stems, branches and petioles can also be affected. Lesions are generally

circular to elliptical with concentric rings. Expanded lesions can girdle stems and

 

 

 

16

weaken or wither apical portions of the tissue. Fruit are affected primarily at the stem
end of the fruit inciting stem end rot (Horsfall & Heurberger 1942). The fungus
appears to invade fruit from the calyx or pedicle and radiates through the stem end of
the fruit to form concentric rings. The infected area is dry, leathery and ﬁrm and may
be covered by a velvety mass of black spores (Jones et al. 1991; Sherf & MacNab
1986). Numerous small (<3 mm) black lesions on fruit and not restricted to the stem

end have also been associated with A. solani (Thomas 1944).

ii. CAUSAL ORGANISM
A. solani is classiﬁed in the subdivision Deuteromycotina (the imperfect fungi),

class Hyphomycetes, order Hyphales (Agrios 1988). A teleomorphic stage (Pleospora
solam' sp. nov.) has been reported (Esquivel 1984) but apparently is not common.
Susceptible hosts, in addition to tomato and potato, include eggplant (Solarium
melongena L.) and various solanaceous weeds such as horse nettle (Solanum
carolinense) and black nightshade (Solanum nigrum L.) (Sherf & MacNab 1986;
Rands 1917). A. solani expresses considerable variability in morphology, physiology
and pathogenicity from one isolate to another (Bonde 1929; Henning & Alexander
1959; Wellman 1943). Although the presence of physiological races has been
suggested, no differential host lines are known to substantiate this. The genetic
variability of the pathogen, and ultimately how an understanding of the variability may
impact disease management programs, is virtually unknown. Petrunak and Christ
(1992) studied protein polymorphism using isozyme analysis and demonstrated a
country (USA) wide sarnpling of A.Solani isolates were distinct from A. alternata

isolates collected from the same host (tomato or potato). The percentage of

 

 

 

 

l7

polymorphic loci detected in the Alternaria isolates examined was 92%, considerably

greater than levels observed for other fungi (Petrunak and Christ 1992).

iii. DISEASE CYCLE AND EPIDEMIOLOGY

A. solani is a multicyclic disease. The pathogen is able to overwinter in soil in
association with plant debris as mycelium, conidia or chlamydospores (Basu 1971;
Patterson 1991). Inoculum can persist on the surface or buried in the absence of host
residue (Basu 1971). In Wisconsin, Rands (1917) demonstrated overwinter survival of
the fungus in infected leaves and survival increased with depth of burial. Patterson
(1991) noted incidence of collar rot decreased with increased inoculum depth. The
relative persistence of inoculum is not known but chlamydospores are thought be the
most important means of survival of primary soilbome inoculum (Patterson 1991).
However, the pathogen’s association with weeds (Rands 1917), seed (Moore et al.
1943) and transplants (Moore 1942) cannot be disregarded as sources of initial
inoculum.

Spore germination occurs within 1 hr under optimum conditions. Free water or
a relative humidity greater than 92% is required for germination (Stevenson &
Pennypacker 1988). Penetration is direct, through wounds or stomata and occurs
within 6 to 12 hr (Waggoner & Horsfall 1969). Infection increases with temperatures
between 12 and 25° C (Moore 1942; Pound 1951). Lesions expand on mature tissue
when the tissue is wet (Waggoner & Horsfall 1969). Infection efﬁciency increases
with the load of fruit on the plant (Horsfall & Heuberger 1942; Waggoner & Horsfall
1969). Sporulation does not begin until lesions attain a size of 3 to 4 mm in diameter

(Rands 1917 ). Corridiophore development occurs in the presence of free moisture and

_.

 

 

18

is triggered by light (Waggoner & Horsfall 1969). Spore formation is favored on
leaves ﬁrst exposed to a dry light period and then a dark wet period (Rotem & Bashi
1969). The diurnal periodicity results in spore release and dissemination during the day
if wind speed is sufﬁcient (Harrison et al. 1965b; Rotem 1964). Somewhat
paradoxically, spore germination and survival is favoured by the dark and inhibited by
light (Stevenson & Pennypacker 1988; Rotem et al. 1985). Spore dispersal peaks soon
after ﬁrst lesions are detectable in mid to late July (Harrison et al. 1965b; Madden et
al. 197 8). Few spores are present prior to this peak and spore numbers are variable
after this peak Lesions can produce up to four crops of spores (Rand 1917) and under
optimum conditions, 5 to 7 days are required from inoculation to production of spores.

In addition to environment, early blight progress is favored by low nitrogen

(Thomas 1948; Horsfall & Heuberger 1942), early host maturity (Barratt & Richards
1944), nematode populations (Barker 1972), wounding (Rotem 1965), soil moisture
stress (Rotem 1969) or other forms of stress associated with enhanced maturation or
senescence of tissue.

In summary, A. solani effectively overwinters in temperate climates or is
introduced to incite foliar epidemics. Disease initiates on mature tissue. Temperatures
between 13 and 27°C and leaf wetness or high RH favor spore germination,
penetration, lesion expansion and sporulation. Dry windy conditions favor dispersal.
Factors that enhance host maturity or senescence enhance disease incidence.

iv. CONTROL

Despite an extensive literature reporting research results on the epidemiology,

etiology and control of the pathogen, early blight is one of the primary diseases of

 

-n

 

 

 

l9

tomato targeted in routine fungicide applications in the northcentral production region.
Extensive surveys and subsequent work have identiﬁed sources of resistance to A.
solani (Alexander et al. 1942; Barksdale & Stoner 1977; Gardner 1988; Maiero et al.
1990a; Nash & Gardner 1988a). However, inheritance of resistance is complex (Nash
& Gardner 1988b; Maeiro et al. 1989). No commercial cultivars, processing nor fresh
market, have acceptable levels of resistance to early blight. O’Leary (1985) has shown
that resistance in some of the most promising lines is a form of horizontal resistance
or rate reducing resistance govemed by the interaction of infection efﬁciency, lesion
area, latent period, sporulation capacity per lesion and sporulation capacity per unit
area.

O’Leary (1985) has demonstrated that levels of resistance can be combined
with a reduced fungicide spray program to achieve control of early blight.

Basu (1974) fumigated infested soil and decreased initial levels of early blight
but growth and yield of tomato plants was not affected. A 2 to 3 year rotation to
decrease initial inoculum has also been suggested (Horsfall & Heurnerger 1942; Rands
1917; Sherf & MacNab 1986) but limited experimental evidence is available to assess
the impact of rotation.

Nitrogen applications, up to a certain optimum, delay plant maturity and reduce
incidence of early blight (Fischer 1986; Horsfall & Heurberger 1942; Jones & Jones
1986; Thomas 1948). Delayed senescence associated with nitrogen appears to decrease

the apparent infection rate of disease progress (Fischer 1986; Mackenzie 1981). Mulch
around plants can have a positive affect depending on the year (Fischer 1986).
The potential for biological control has received even less attention than

cultural practices. Brame and Flood (1983) demonstrated a 2-day pre—incubation with

—.

 

 

 

 

20

Aureobasidium pullulans on leaf surfaces signiﬁcantly reduced infection and growth of
A. solani. However, the antagonism could not be associated with inhibitory
metabolites. Rather, A. pullulans incited host defense responses and this impacted
infectivity of A. solani (Flood & Rees 1986). Casida and Lukezic (1992) demonstrated
a Pseudomonas isolate, strain 679-2, provided a reduction in the severity of early
blight in ﬁeld trials. No adverse affects to the plant were noted and antagonism was
associated with a water soluble inhibitory compound. Leben and Daft (1965) spray
inoculated tomatoes with a bacteria, originally isolated as an epiphyte on cucumbers,
and reduced early blight incidence when challenged 2 -3 days later.
Multiple disease control strategies have been researched but few have
progressed sufﬁciently far to offer adequate control of early blight. Fungicides

continue to be the primary and often only strategy for control of early blight.

b. ANTHRACNOSE
A fungus associated with anthracnose was ﬁrst described in 1879 by Saccardo
(Sherf & MacNab 1986). Anthracnose has been recorded in Asia, Europe, Africa, the
East Indies and North America (Jones et al. 1991). In North America, anthracnose is
an economic problem in the northeast and rnidwest USA. and central Canada. The
literature has recently been reviewed by Dillard (1992).

Anthracnose is primarily a disease of ripe fruit and is the most important fruit
rot disease of processing tomatoes in the northcentral production region (Barksdale &
Stoner 1981; Preucher et al. 1992; Stevenson et a1. 1978). Disease incidence can be as
high as 70% (Wilson & Runnels 1949) in the absence of control and 5 to 15 % even

with repeated applications of fungicide (Sherf & MacNab 1986). Amonut of disease is

-4»

 

 

 

 

 

21

a function of the production system. Harvest of ripe fruit for processing is often
delayed for extended periods to allow optimum timing for once-over machine
harvesting. In contrast, fresh market tomatoes are picked in multiple harvests at the
breaker stage (ﬁrst appearance of color) and are shipped and often consumed before

latent infections develop into lesions.

i. SYMPTOMS

Symptoms appear only on on ripe or senescent fruit (Dillard 1992; Jones et al.
1991; Scherf & MacNab 1986). Lesions on ripe fruit ﬁrst appear as light brown ﬂecks
and expand as sunken circular lesions up to 12 mm in diameter. Tissue in the center of
the lesion darken and contain acervuli that bear masses of salmon-colored spores
during wet weather. Large portions of the fruit become rotted as lesions expand and
coalesce and secondary organisms invade the tissue.

Leaves, stems and roots may also be affected. Leaf symptoms are rare but can
appear 5 to 7 days after inoculation and enlarge to a maximum size of 2 mm. Leaf
lesions are sunken, necrotic in the center and may be surrounded by a halo (Younkin
& Dirnock 1944). Root symptoms include black dots (microsclerotia of the fungus),

brown lesions or brown root rot.

ii. CAUSAL ORGANISM

Colletotrichum coccodes (Wallr.) Hughes is the most common species
associated with anthracnose fruit rot but numerous other species can infect tomato fruit
(Batson and Roy 1982; Stevenson et al. 1978). Black dot disease of tomato roots is

also associated with C. coccodes but is not a known concern in the northcentral

 

 

 

 

 

22

production region.

C. coccodes is classiﬁed in the subdivision Deuteromycotina (the imperfect
ﬁmgi), class Coelomycetes, order Melanconiales (Agrios 1988). C. coccodes isolates
vary considerably in pathogenicity, growth rates, pigmentation, sclerotia Size and in
other characteristics (Dillard 1992; Sherf & MacNab 1986). No details of population
diversity is known. C. coccodes has a wide host range including members of the
Solanaceae, Leguminaceae and Cucurbz'taceae family. Over 19 families comprising 68

species, including numerous weeds, have been identiﬁed as hosts (Jones et al. 1991).

iii. DISEASE CYCLE AND EPIDEMIOLOGY

Inoculum inciting fruit rot may originate from overwintered crop debris,
alternative hosts or secondary inoculum from other infected plant tissue on other
portions of the plant.

Acervuli on infected tissue give rise to sclerotia, the primary mechanism of
overwintering and survival for the ﬁmgus (Dillard 1992). Inoculum persists in
association with overwintering tomato skin tissue (Dillard 1990; Farley 1972). Dillard
(1990; 1992) buried colonized skins of tomato fruits 0, 10 and 20 cm deep and after 3
years found 70% of tissue harbored viable propagules and 90% of the sclerotia were
viable.

Weeds or other crops can serve as hosts and as a source of primary inoculum
to susceptible crops (Batson & Roy 1982; Raid & Pennypacker 1987). Komm &
Stevenson (197 8) observed incidence of C. coccodes propagules and disease incidence
on potato was considerably less in a reclaimed forest land as compared to a ﬁeld with

a history of potato production. However, virgin forest soils did harbor inoculum and

 

 

 

 

23

incite disease. Inoculum levels increase in the absence of rotation.

Younkin & Dimock (1944) experimentally demonstrated that C. coccodes is
able to infect foliage. Leaf lesions on attached leaves can sporulate but not abundantly
(Y ounkin & Dimock 1944). Farmer (1959) and Illrnan et al. (1959) demonstrated
detached and senescent tissue supported profuse colonization and sporulation. Pantidou
and Shroeder (1955) reported leaves can be infected, especially lower leaves, in the
ﬁeld and provide secondary inoculum to fruit. Wounding (sand-blasting) and extended
post-inoculation wetting periods signiﬁcantly increase infection and lesion
development on potato foliage (Johnson & Miliczky 1993). The relative importance
and relationship of foliage infection, importance of foliage as a source of secondary
inoculum, the environmental conditions favorin g leaf infection and ﬁnal incidence of
tomato fruit rot, is largely unknown. Work is currently in progress to address these
issues (Hausbeck & Linderman 1992).

Growth from sclerotia is optimum at 28°C (Dillard 1988) and favored by wet
conditions. Sclerotia germinate by producing hyphae or by producing conidia in
acervuli on the sclerotia surface.

Inoculum may come in contact with fruit resting on the soil or be splashed by
rain onto susceptible surfaces. Fruit in various stages of development are susceptible to
direct penetration in the absence of wounding. Infections of green fruit are latent
(Farmer 1959; Fulton 1948) and as the fruit ripens, symptoms develop rapidly.
Symptoms develop rapidly on inoculated ripe fruit. Gemrination of conidia occurs at
an optimum temperature of 22°C and a minimum of 10 hr of continuous wetness is
required for infection. Lesion expansion is favored by temperatures 16 to 31°C and

subsequent conidia formation increases with increasing temperature from 16 to 28°C

 

 

 

 

24

(Dillard 1988; 1989).

Deterrninate and open growth, early maturity and early defoliation has been
associated with increased levels of anthracnose on fruit (Wilson & Runnels 1949).
Barksdale & Koch (1969) demonstrated soil type impacts disease incidence. They
recorded almost twice the general amount of natural infection on plants grown in
' sandy soil as compared to those grown in clay soil.

In summary, C. coccodes is associated with numerous hosts, including weeds
common in the northcentral production region. Sclerotia can persist in association with
host debris for 3 or more years and is the primary source of initial inoculum. Disease

progress is favored by warm temperatures (24 - 28°C) and wet conditions.

iv. CONTROL

Control recommendations include crop rotations of 3 to 4 years that exclude
known susceptible hosts (e.g. potatoes), adequate control of weed hosts, timely harvest
of ripe fruit, use of tolerant genotypes and routine applications of fungicides.

Few studies have been conducted to determine the impact of crop rotation and
weed management programs. Precise timing of machine harvesting and the use of
ethephon to speed up ripening help reduce anthracnose levels (Sherf & MacNab 1986).

Genetic resistance is available in advanced determinate breeding lines that have
yield and fruit qualities Similar to commercial lines (Barksdale & Stoner 1981).
Breeding lines exhibited 87 - 99% less anthracnose during natural infection as
compared to a susceptible control (Stevenson et al. 1978). Although resistance is
controlled by a number of genes, Miller et al. (1984) concluded relatively rapid

genetic advance should be possible in breeding and selection of resistant genotypes.

 

 

 

 

25

Anthracnose resistance incorporated in the advanced breeding lines offered control

comparable to susceptible controls but with 3 to 7 fewer fungicide sprays (Barksdale

& Stoner 1981).

Breeding for disease resistance is complicated by the ability of multiple species
able to incite anthracnose fruit rot and the variability of virulence among isolates
within each species (Batson & Roy 1982). However, resistance to one species may be
correlated with resistance to other species (Barksdale 1972; Stevenson et al. 1978).
The relative importance of the various species in ﬁeld incidence of anthracnose is not
known.

Currently, anthracnose is controlled through the routine application of
protective fungicides. Because the anthracnose fungi can infect green fruit, ﬁmgicide
recommendations call for an initial spray when fruit ﬁrst set.

Anthracnose was reduced considerably with the application of 14 pounds/acre
of maneb (Dithane M—22) directly to the soil in late June. Three foliar sprays of
maneb during the season enhanced control (Crossan et al. 1963).

Leben and Daft (1965) evaluated the potential of biological control. They spray
inoculated tomatoes with a bacteria, originally isolated as an epiphyte on cucumbers, 2

days prior to a challenge inoculation with the anthracnose fungus. Anthracnose

incidence was reduced.

c. RHIZOCTONIA SOIL ROT

Rhizoctonia solani was ﬁrst observed as a pathogen of potato in 1858 by Julius
Kuehn. Rhizoctonia appears to have an unlimited host range and is studied in hundreds

of research programs (Parmeter 1970). The pathogen attacks tomato worldwide and is

 

 

 

 

 

 

 

26

able to incite damping off, root rot, stem canker and fruit rot. In ﬁeld production
systems, Rhizoctonia soil rot is the principal component of the soil rot disease
complex of tomato fruit and can result in losses up to 75% (Batson 1973; Jones &

McCarter 1974).

i. SYMPTOMS

Soil rot may affect fruit at any stage of development but is most common on
ripening fruit at or near the soil. Lesions commence as small, ﬁrm brown spots that
progressively enlarge. Lesions expand rapidly, may have concentric zones and become
soft and mushy as the pathogen rarniﬁes through the tissue and secondary organisms
move in. The pathogen may also cause damping off or poor plant productivity of

young plants early in the season.

ii. CAUSAL ORGANISM

Rhizoctonia solani Kuehn is a heterogenous collection of strains that vary
considerably in pathogenicity, culture characteristics and saprophytic ability. The
species has sterile mycelia (no spores) and is generally divided into subgroups based
on anastomosis behavior. The pathogen is ubiquitous and able to attack most plant
species. It is classiﬁed in the subdivision Deuteromycotina (the imperfect fungi), class
A gonomycetes (Mycelia Sterilia), order A gonomycetales (A grios 198 8). The

teleomorph is classiﬁed in the subdivision Basidiomycotina.

 

5'5

 

 

 

 

 

 

 

 

27

iii. DISEASE CYCLE AND EPIDEMIOLOGY

Papavisas et al. (1975) demonstrated in Maryland that saprophytic activity of R
solani peaks in the top 10 cm of the soil and soon after soil incorporation of crop
residue. Soil populations declined rapidly, following conventional tillage practices, to
relatively low numbers by the following spring. R. solani is highly dependent on plant
tissue and disappears with the reduction of food bases (Papavisas et a1. 1975).
Persistence in the soil is dependent on the strain (Carling and Leiner 1990; Parrneter
1970). Penetration is direct with an optimum temperature for infection of 25°C and a
requirement for free moisture or high relative humidity (Gonzalez & Owen 1963).

Once successful invasion occurs, lesion expansion is not limited by moisture.

iv. CONTROL

Control recommendations include staking fresh market tomato varieties or
using plastic or paper mulch (Jones & McCarter 1974) to limit fruit contact with soil.
Staking is labor intensive and recent reports indicate a move away from this
horticultural practice. Straw mulch reduces soil rot but affects are variable with
location (Jones & McCarter 1974). Staking or use of mulch is not practical in
processing tomato production.

Polygenic resistance, useful for processing cultivars, has been identiﬁed
(Barksdale 1974).

Fungicide applications at regular intervals during fruit development can be
moderately effective in providing control (McCarter & Barksdale 1977) but are not
considered to be economical for fruit rot control (Jones et a1. 1991). Alternatively,

Crossan et al. (1963) showed soil rot was reduced considerably with the application of

 

 

 

ﬂ»— ’1’

28

14 pounds/acre of maneb (Dithane M-22) directly tO the soil in late June. Three foliar
sprays of maneb during the season did not increase disease control. The economics of
the later approach is questionable (McCarter and Barksdale 1977) and is not practiced.
Because the pathogen survives in colonized plant debris, it is inﬂuenced more

by tillage practices than many other soilbome pathogen (Sumner et al. 1986a). The
pathogen does not persist well at depths of 5 - 10 or more cm (Papavisas et al. 1975).
Moldboard plowing and burial of crop debris has greatest beneﬁt where high inoculum
levels of R. solani exist (Sumner et al. 1986a). Root disease severity is probably
inﬂuenced more by the previous crop however, than by tillage system (Sumner et al.
1986b; Rush & Winter 1990).

Lewis et al. (1990) evaluated the potential to biologically control R. solani with

 

a T richoderma and Gliocladium isolate. After 5 years of greenhouse and ﬁeld

 

evaluation, the authors concluded some control was possible in the greenhouse but this
form of biological control was not useful in ﬁeld production. Limitations to successful
control included the ubiquitous nature of R. solani having broad ecological

capabilities, extended periods of time associated with disease progress and the

 

complexity of environmental, physical and biological factors that impacted pathogen—

 

biocontrol agent interactions.

(1. FUNGAL FOLIAR-FRUIT PATHOSYSTEM2SUMMARY

The epidemiology, etiology and biology of early blight and anthracnose has
not been systematically compared. However, a review of the literature demonstrates
that each disease can be endemic and is favored by similar environmental conditions

and host maturity. The life cycle and epidemiology of R solani differs considerably

 

 

29

but is also endemic and the target of routine ﬁmgicide applications.

2. THE BACTERIAL PATHOSYSTEM
a. BACTERIAL CANKER, BACTERIAL SPOT AND BACTERIAL SPECK
Bacterial canker, bacterial spot and bacterial speck, are economically important
bacterial diseases throughout the northcentral production region. Each disease occurs
yearly but sporadically, not occurring on every farm every year, and is differentially
affected by the environment and crop production system. However, the pathogens that
cause canker, speck and spot share many biological and epidemiological features and
comprise a pathosystem.

Bacterial canker, spot and speck were ﬁrst reported in 1910, 1920 and 1933,

 

respectively (Bryan 1933; Doidge 1921; Smith 1910) and occur worldwide (Jones et

 

al. 1991; Sherf and MacNab 1986). Canker is one of the most destructive diseases of
tomato but each disease can be responsible for up to 70% loss in yield or fruit quality
(Pohronezny'and Volin 1983; Strider 1969; Sherf and MacNab 1986; Yunis et al.
1980). Marketable yield loss is associated with defoliation and fruit lesions with all

three diseases and early season wilt in the case of canker. Lesions on fruit limit sales

 

of fresh market tomato and hinder skin removal and product quality of processing

tomato.

i. SYMPTOMS
A wide array of symptoms, that can be categorized as two separate phases, are
associated with bacterial canker (Gleason et al. 1993; Strider 1969). A systemic phase,

is initially manifested early in the season as unilateral wilting of leaves but eventually,

 

 

 

30

the entire plant may wilt or develop stem cankers and die. A second phase is
diagnosed initially as ﬁring of leaf margins. Necrosis and wilting of entire stems ensue
as bacteria basipetally migrate into the tissue. Fruit lesions are raised brown spots
often surrounded by a yellow halo giving the appearance of a ”bird’s eye”, a diagnostic
feature of canker.

Spot may incite blighting of leaves associated with coalescing of numerous
lesions. All above ground parts of the plant can be affected and individual lesions are
dark and rarely larger than 3 mm. Fruit spots are 2 to 10 mm in diameter, initially
appearing as small water soaked areas but later turning brown to gray. Subepidermal
infections result in a scabby appearance as surface tissue disintegrates. Speck, like
spot, is able to affect all above ground portions of the plant. Lesions are small, no
larger than one mm. Many lesions may occur on infected tissue and coronitine, a
phytotoxin produced by the speck pathogen, dissipates to incite large areas of
chlorosis. Superﬁcial and slightly protruding black lesions form subepidermally on

fruit. Lesions may coalesce and affect large expanses of the fruit.

ii. CAUSAL ORGANISM

Bacterial canker is caused by Clavibacter michiganensis subsp. michiganensis
(Smith) Davis et al. (Cmm), a gram positive, non-spore forming pleomorphic
bacterium. Differences in pathogenicity have been reported (de Vries 1990) but the
genetic diversity of the pathogen is not known.

Bacterial speck is caused by Pseudomonas syringae pv. tomato (Okabe) Young;
Dye & Wilkie (Pst), a motile gram negative rod. The pathogen appears to be

comprised of two closely related lineages (Cooksey & Graham 1989; Denny et al.

 

 

 

 

 

 

31

1988). Two races have been identiﬁed based on virulence for differential hosts
(Lawton & McNeil] 1986).

Xanthomonas campestris pv. vesicatoria (Doidge) Dye (Xcv), a motile, gram
negative rod shaped bacterium, incites bacterial spot on tomato and pepper. The
pathovar is phenotypically (Dye et al. 1964), serologically (Jones et al. 1993b),
pathogenically (Minsavage et al. 1990) and genotypically diverse (Vauterin et al.
1990). The pathovar has been described with the ability (Dowson 1949), general
inability (Gitaitis et al. 1987) or variable ability (Dye 1964) to hydrolyse starch. In
North America, primarily Georgia and Florida, where most research on Xcv occurs, a
starch negative reaction is considered diagnostic (Gitaitis et al. 1987). Starch positive
strains associated with tomato plants in Georgia, have been shown to be non-
pathogenic opportunistic epiphytes (Gitaitis et al. 1987). Likewise, pectolytic and non-
pectolytic isolates are known to exist. Beaulieu et al. (1991) concluded pectolytic
activity was correlated with the geographical origin of isolates. For example, 90% of
isolates obtained from Argentina were pectolytic as compared to only 1 of 374 isolates
originating from the United States.

Minsavage et al. (1990) categorized Xcv isolates into groups and races based
on virulence for pepper and tomato genotypes. The pepper group was subdivided into
3 races. Race 1 has virulence for pepper only and race 2 and 3 have virulence for
tomato plants and selected pepper genotypes. Typing of pepper races is based on
differential reaction on near isogenic pepper lines with an interaction that functions in
a gene-for-gene manner. Minsavage et al. (1990) designated a tomato group of strains,
comprised of one race (Tl), based on ability to infect tomato genotypes and no known

pepper genotypes. More recently, a second tomato race (T2) has been identiﬁed (Wang

 

 

 

32

et al. 1990). T2 strains, also designated ”B” strains, are pectolytic and/or starch

hydrolytic and serologically distinct as compared to T1 or ”A” strains (Jones et al.

1993).

iii. DISEASE CYCLE AND EPIDEMIOLOGY

Cmm, Pst and Xcv are able to overwinter in association with crop debris,
weeds and volunteer plants (Chang et al. 1992; Gleason et al. 1991; Jardine et al.
1988; Peterson 1963). Overwintered inoculum may serve as a source of inoculum to
subsequent tomato crops (Chang et al. 1992; Gleason et al. 1991; Jardine et al. 1988).
The bacteria are poor saprophytes and do not persist in soils for long periods of time
in the absence of host debris (Gleason et al. 1991; Jones et a1. 1991). Overwintered
sources of inoculum appear to lead to epiphytic populations on tomato leaf surfaces
and disease symptoms appear mid to late season after a threshold of 10° cfu per leaﬂet
is achieved (Gleason et al. 1991). All three bacteria are known to be seedbome
(Dhanvantari 1989; Gardner and Kendrick 1921; McCarter et al. 1983; Sijam et a1.
1991). Contaminated seed is the most important source of inoculum (Dhanvantari

1989) for bacterial canker. Seed borne inoculum gives rise to systemically infected

 

seedlings that often remain symptomless for up to 8 wks after ﬁeld setting. Wilt and
symptoms of primary canker are enhanced by stress conditions such as low moisture.
The importance of Pst inoculum on seed is supported by a worldwide outbreak
of the disease that occurred in 1978 (Goode and Sasser 1980). In the case of Xcv, the
relative importance of seedbome inoculum, especially in the northcentral region, is
unknown. Using current screening procedures, the frequency of contaminated tomato

seedlots is reportedly low.

 

 

 

 

33

Secondary spread of all three bacteria occurs by handling of transplants prior to
ﬁeld setting, by splash dispersal, insects and ﬁeld production activities (Bashan 1986;
Chang et al. 1991; McCarter et al. 1983; McInnes et al. 1988; Volcani 1969).
Optimum disease progress is enhanced by wet conditions and temperatures of 25° to
32°, 18° to 24° and 24° to 30°C for Cmm, Pst and Xcv, respectively (Jones et al. 1991;
Sherf and MacNab 1986). Infection may occur through stomata but is favored by
wounds inﬂicted by insects, cultural practices, wind or sandblasting damage (Getz et
al. 1983b; Carlton et a1. 1992; Vakili 1967). Fruit infection occurs through natural

wounds and when fruit is less than 3 mm in diameter (Getz et al. 1983a).

iv. CONTROL

Prevention is the most important control strategy for controlling bacterial
canker, speck and Spot (Goode and Sasser 1980; Gitaitis et al. 1992). Zero tolerance in
seedlots is the goal of the industry but has proven unrealistic due to the limits of
detection and seed treatment technologies (Sasser and Goode 1980). For example,
Cmm seed infection levels less than 0.1% are difﬁcult to detect using current seed
plating assays (Dhanvantari 1989) but as few as 0.01% infected seedlings, grown
according to speciﬁc cultural practices, can initiate a serious epidemic in tomato
plantings (Chang et al. 1991; Gitaitis and Beaver 1991).

Currently, samples of tomato seeds are ground and the extract plated on
selective media (Gitaitis et al. 1992). Suspect colonies are evaluated by selected
phenotypic tests, ELISA, fatty acid proﬁle analysis, or induction of a hypersensitive
response on a non-host (Gitaitis et al. 1992). Each presumptive diagnosis is followed

by pathogenicity tests. No protocols are available for rapid, non-presumptive

 

 

 

 

 

34

identiﬁcation of the bacteria. Diagnostic probes speciﬁc for Pst (Cuppels et al. 1990)
and Cmm (Thompson et al. 1989) have been developed but not commercially
deployed.

Sanitation in greenhouse production of transplants and around the ﬁeld (e. g.
removal of weed hosts and volunteers) is important (Sherf and MacNab 1986). Crop
rotations are routinely recommended (Jones et al. 1991). Gleason et al. (1991) and
Jardine et al. (1988) have demonstrated burial of infected host debris enhances decline
of overwintered inoculum. Thus, fall plowing is also often recommended. Other
cultural practices include the use of windbreaks to decrease sand blast injury, and
avoidance of activities that wound tomato plants.

Chemical based control generally has not proven effective though often used.
Jardine and Stephens (1987) demonstrated applications of bactericides were effective
only when conditions for disease were limiting. Yunis et al. (1980) applied copper
sprays weekly and reduced speck disease severity. However, routine use of
streptomycin or copper has led to bacteria populations resistant to the chemicals
(Bender and Cooksey 1986; Marco and Stall 1983; Stall and Thyer 1962). Bactericides
must be applied within 24 - 48 hrs post infection and on a 4 to 7 day schedule if they
are to have any utility (Jardine and Stephens 1987). Routine fungicide applications do
not control the bacteria (MacNab 1980).

No commercial cultivars have acceptable levels of genetic resistance despite
extensive surveys of domesticated and wild germplasm (Alexander 1942; Crill et al.
1972; Lawson and Summers 1984a & b; Pilowsky and Zutra 1982; Scott and Jones
1986; Thyr 1968). Resistance to Cmm is complex and not easily transferred to

commercial cultivars (Jong and Honma 1976). Pitblado and Kerr (1979) identiﬁed a

 

l

 

 

35

tomato genotype with vertical resistance (PtO gene) to Pst. However, Lawton and
McNeill (1986) demonstrated a virulent race was present at low levels in natural
populations even before the resistance gene was deployed. Likewise, Scott and Jones
(1986) identiﬁed a tomato genotype, Hawaii 7998, with resistance to Xcv. However,

before Hawaii 7998 was generally deployed, virulent strains from Argentina were

identiﬁed (Wang et al. 1990).

b. SUMMARY OF BACTERIAL PATHOSYSTEM

Bacterial canker, spot and speck are important diseases in the northcentral
production region. Cultural practices, including crop rotation and routine chemical
sprays have not prevented marketable yield-reducin g epidemics. Genetic resistance is

currently not deployed and sources of resistance have not proven durable or easy to

 

incorporate into commercial cultivars. Prevention is the key to control. However,
detection assays are not sensitive enough to detect epidemiologically signiﬁcant
seedbome populations. Advances in developing speciﬁc detection protocols and

immementing breeding programs have not been informed by an understanding of the

chromosomal based genetic diversity of each pathogen.

D. ASSESSING POPULATION GENOTYPIC DIVERSITY OF PLANT

PATHOGEN S

Chromosome based assessment of genetic diversity has been limited by
technology. Recently, multilocus enzyme electrophoresis (MLEE) (Denny et al. 1988),

restriction enzyme digestion of total DNA (Cooksey and Graham 1989; Hartung and

 

 

36

Civerelo 1987), and restriction fragment length polymorphism (RFLP) analyses with
speciﬁc probes (Berthier et al. 1993; Denny et al. 1988; Leach et al. 1992; Levy et al.
1991) have been utilized to assess genetic diversity of plant pathogens. Alternatively,
polymerase chain reaction (PCR) based protocols: using arbitrary primers (Welsh and
McClelland 1990; Williams et al. 1990), primers corresponding to t-RNA (Welsh and
McClelland 1991), or primers corresponding to 168 and 23S genes (Jensen et al. 1993)
have been used to discern difference among strains of bacteria. The potential of each
protocol to delineate genotypic diversity of plant bacteria has not been established.
This thesis explores the potential of yet another PCR-based approach, known as
rep-PCR and recently reviewed by Lupski and Weinstock (1992). Families of
repetitive sequences are dispersed throughout the genome of diverse bacterial species

(V ersalovic et al. 1991; Koeuth et al. 1994). Three families, though not related by

 

 

DNA sequence homology, include the 3540 bp repetitive extragenic palindromic
(REP) sequence (Gilson et al. 1984; Higgins et al. 1982), the 124-127 bp
enterobacterial repetitive intergenic consensus sequence (ERIC) (I-lulton et al. 1991;
Sharples and Lloyd 1990) and the recently discovered 154 bp BOX elements (Martin
et al. 1992). Primers corresponding to the repeated palindromic sequences anneal to
DNA via PCR and DNA between two adjacent sequences is ampliﬁed according to the

processing limits of the Taq polymerase enzyme. PCR products are separated on

 

agarose gels and provide species and strain speciﬁc banding patterns (V ersalovic et al.
1991; de Bruijn 1992; Koeuth et al. 1993).

The relative potential of rep-PCR to assess genotypic diversity has not been
fully determined. Patterns of similarity in rep-PCR banding patterns correspond to

phylogenetic relationships determined by MLEE (de Bruijn 1992) and RFLP analyses

 

 

 

 

37

(Judd et al. 1993).

In the case of MLEE, variation in mobility of proteins can be directly
associated with alleles of known genes. Variation appears to be selectively neutral, and
can be scored to statistically determine genetic diversity among bacterial isolates and
phylogenetic relationships among lineages (i.e.clones) can be ascertained (Selander and
Musser 1990). In contrast, polymorphism in rep-PCR patterns can not be equated to
Single loci nor can such polymorphisms be assumed neutral. To the contrary, rep-like
sequences may have a functional role (Lupski and Weinstein 1992) and are highly
constrained over time within pathogenic clones (Woods et al. 1992). Rep-like
sequences may in fact have a role in genome organization and niche specialization
(Kraweic 1985). A theoretical framework for determining genetic distance and

evolutionary structure of bacteria using rep-PCR has not yet been developed.

 

 

Thorough sampling of genotypic diversity of clinical and animal pathogens has
provided a framework to assort pathogenic populations with respect to host species,
geographic distribution and nature of disease caused (Selander and Musser 1990).
Likewise, proposed disease speciﬁc virulence factors can be associated with speciﬁc
clonal groups and disease speciﬁcity (Achtrnan and Pluschke 1986; Selander and
Musser 1990). Similar sorting of bacteria may be possible using rep-PCR and may be
useful for identifying true pathogenic variants and elucidating the diversity of bacterial
pathogens and symbionts (de Bruijn 1992; Judd et al. 1993; Versalovic et al. 1993;
Woods et al. 1992; Appendix D). The implication, similar to the case of pathogens of
humans and animals (Selander and Musser 1990), is that the unit important to devising
integrated disease management strategies to control plant pathogenic bacteria, is not

the species, subspecies or pathovar, but the clone. A clone, or evolutionary lineage,

 

 

38

may or may not be coincident with taxonomical divisions.

A key question that plagues plant pathologists concems the origin of
pathogenic variants and the factors that govern host range. Dr. Gabriel's group
(Swarup et al. 1991; 1992; Waney et al. 1991) have shown host-speciﬁc virulence
(hsv) genes can frmction as positive factors to determine host range. Horizontal
transfer (i.e. a recombinational mechanism of convergence) of hsv gene(s) could give
rise to clonal groups with distinctive chromosomal genotypes but similar host range. In
contrast, Stall et al. (1994) suggest that host range, for example in Xcv, ”is determined
by avirulence genes carried on plasmids”. Kearney et al. (1988) suggest mutation of
single genes give rise to virulent pathotypes. Indeed, avirulence genes cloned into
foreign backgrounds alter host range (Kobayashi et al. 1989; Whalen et al. 1988).

However, this may be a gratuitous function (Gabriel 1989) and a result of common

 

 

ancestry of the pathogen or host (Heath 1991).

Emphasis on single genes and gene-for—gene models has been associated with a
boom-and-bust cycle in genetic resistance breeding. Pathogenic variants, for example
within Xcv, have traditionally been described in terms of race governed by single
genes for avirulence that can be identiﬁed if genetic lines within a host species have
differential sets of genes for resistance (Minsavage et a1. 1990). However, avirulence

genes may not necessarily be a component of basic compatibility (Heath 1991). This

 

thesis attempts to use protocols that describe pathogenic diversity in terms of overall
chromosomal organization using rep-PCR, and identify lineages or clones, primarily in
Xcv, that may have arisen as a function of basic compatibility with tomato.
Assessment of chromosomal organization has been useful to resolve pathotypes in

other pathosystems (Leach et al. 1992; Levy et a1. 1991). The potential to capitalize on

 

 

 

 

39

knowledge of genetic diversity has only begun to be explored and may provide a
framework for understanding pathogenesis, evolutionary dynamics and optimal
methods for the implementation of integrated disease management strategies, including

the deployment of host resistance (Leach et al. 1992; Levy et al. 1991).

 

 

 

 

 

4O

LITERATURE CITED

Achtman, M. and G. Plushke. 1986. Clonal analysis of descent and virulence among
selected Escherichia coli. Ann. Rev. Microbiol. 40:185-210.

Agrios, G.N. 1988. Plant Pathology. 3rd edition. p.803. Academic Press Inc., NY.
Alexander, LA, and V.W. Wright. 1942. A survey of the genus Lycopersicon for
resitance to the important tomato diseases occurring in Ohio and Indiana. Plant Dis.
Rep. 136:51-85.

Allen, R, D. Van Dusen, J. Lundy, and S. Gliessman. 1991. Integrating social,
environmental and economic issues in sustainable agriculture. Amer. J. Alt. A gr.

6:34-39.

Armburst, D.V., J.D. Dickerson, and J.K. Greig. 1969. Effect of soil moisture on the
recovery of sandblasted tomato seedlings. J. Amer. Soc. Hort. Sci. 94:214-217.

Barker, KR. 1972. Correlations of initial densities of Meloidogyne incognita on tomato
yield and incidence of early blight. Phytopatholgoy 62:801(abstr.).

Barksdale, T. H., and A. K. Stoner. 1981. Levels of tomato anthracnose resistance
measured by reduction of fungicide use. Plant Dis. 65:71-72.

Barksdale, T. H., and E. J. Koch. 1969. Methods of testing tomatoes for anthracnose
resistance. Phytopathology 59:1373-1376.

Barksdale, TH, and AK. Stoner. 1973. Segregation for horizontal resistance to tomato
early blight. Plant Dis. Rep. 57:964-965.

Barksdale, TH. 1972. Resistance in tomato to six anthracnose fungi. Phytopatlrology
62:660-663.

Barksdale, TH. 1974. Evaluation of tomato fruit rot resistance to Rhizoctonia soil rot.
Plant Dis. Rep. 58:406-408.

Barnes, JP, and AR. Putnam. 1987. Role of benzoxazirrones in allelopathy by rye
(Secali cereale L.). J. Chem. Ecol. 13:889-906.

Barnes, J .P., and AR. Putnam. 1983. Rye residues contribute weed suppression in no-
tillage cropping systems. J. Chem. Ecol. 9:1045-1057.

Barratt, R.W., M.C. Richards. 1944. Altemaria blight versus the genus, Lycopersicon.
N.H. Agric. Exp. Stn. Bul. 82. 25pp.

Bashan, Y. 1986. Field dispersal of Pseudomonas syringae pv. tomato, Xanthomonas

 

 

 

 

 

41

campestris pv. vesicatoria, and Altemaria macrospora by animals, pe0ple, birds,
insects, agricultural tools, aircraft, soil particles, and water resources. Can. J. Bot.
64:276-281.

Basu, RB. 1974. Measuring early blight, its progress and inﬂuence on fruit losses in
nine tomato cultivars. Can. Plant Dis. Surv. 54:45-51

Basu, PK. 1971. Existence of chlamydospores of Altemaria porri f.sp. solani as over-
wintering propagules in soil. Phytopathology 61:1347-1350.

Basu, PK. 1974. Reduction of primary infection of tomato early blight by fall
fumigation with Vorlex. Can. Plant Dis. Surv. 54:24-25.

Batson, W. E., and K. W. Roy. 1982. Species of Colletotrichum and Glomerella
pathogenic to tomato fruit. Plant Dis. 66:1153-1155.

Batson, W.,Jr. 1973. Characterization and control of tomato fruit rot. Plant Dis. Rep.
57:453-456.

Beaulieu, C.B., G.M. Minsavage, B.C. Canteros, and RE. Stall. 1991. Biochemical and
genetic analysis of a pectate lyase gene from Xanthomonas campestris pv. vesicatoria.
Mol. Plant-Microbe Interact. 4:446-451.

Bender, CL, and DA. Cooksey. 1986. Indigenous plasmids in Pseudomonas syringae
pv. tomato: conjugative transfer and role in copper resistance. J. Bacteriol.
169:470-474.

Benoit, GR, and MJ. Lindstrom. 1987. Interpreting tillage-residue management
effects. J. Soil and Water Conserv. Mar-Apr :87-90.

Berthier, Y., V. Verdier, J. Guesdon, D. Chevrier., J. Denis, G. Decoux, and M.
Lemattre. 1993. Characterization of Xanthomonas campestris pathovars by rRNA gene
restriction patterns. Appl. Environ. Microbiol. 59:851-859.

Beste, CE. 1973. Evaluation of herbicides in no-till planted cucumbers, tomatoes and
lima beans. Proc. Northeastern Weed Sci. Soc. 27:232—239.

Beste, CE. 1976. An evaluation of no-tillage seeded tomatoes. HortScience 11:298
(abstr).

Bird, G.W. 1993. SARE (Sustainable Agriculture Research and Education Program).
HortScience 28:443 (abstr).

 

Bonde, R. 1929. Physiological strains of Altemaria solani. Phytopathology 19:533-548.

Brame, C., and J. Flood. 1983. Antagonism of Aureobasidium pullulans towards
Altemaria solani. Trans. Br. Mycol. Soc. 81:

 

 

 

 

. i I

42

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

Brown, H.D. 1929. Loss caused by Septoria leaf Spot in the tomato canning crop of
Indiana - 1928. Plant Dis. Rep. 13.:164-165.

Bryan, MB. 1933. Bacterial speck of tomatoes. Phytopathology 23:897-90.

Bugg, R.L. 1992. Using cover crops to manage arthropods on truck farms. HortScience
27:741-745.

Carling, DE, and RH. Leiner. 1990. Virulence of isolates of Rhizoctonia solani AG-3
collected from potato plant organs and soil. Plant Dis. 74:901-903.

Carlton, W.M., M.L. Gleason, and EJ. Braun. 1992. Entry of Clavibacter
michiganensis subsp. michiganensis into tomato plants through hydathodes.
Phytopathology 82:(abstr).

Casida, L.E.,Jr., and FL. Lukezic. 1992. Control of leaf spot diseases of alfalfa and
tomato with applications of the bacterial predator Pseudomonas strain 679-2. Plant Dis.
76:1217-1220.

Chang, R.J., S.M. Ries, and J.K. Pataky. 1991. Dissemination of Clavibacter
michiganensis subsp. michiganensis by practices used to produce tomato transplants.
Phytopathology 81:1276-1281.

 

Chang, R.J., S.M. Ries, and J.K. Pataky. 1992. Local sources of Clavibacter
michiganensis subsp. michiganensis in the development of bacterial canker of tomatoes.
Phytopathology 82:553-560.

Cooksey, D.A., and J.H. Graham. 1989. Genomic ﬁngerprinting of two pathovars of
phytopathogenic bacteria by rare-cuttin g restriction enzymes and ﬁeld inversion gel
electrophoresis. Phytopathology 79:745-750.

Cooley, D. R. 1993. Food and the environment: IPM meets the let century. Plant
Disease 77:296.

 

Coolrnan, R.M., and GD. Hoyt. 1993. The effects of reduced tillage on the soil
environment. HortTechnology 32143-145.

Crill, P., J.P. Jones, and D.S. Burgis. 1972. Relative susceptibility of some tomato
genotypes to bacterial spot. Plant Dis. Rep. 56:504-50.

Crossan, D.F., DJ. Fieldhouse, A.L. Morehart, and J.F. Baniecki. 1963. The effect of
fungicide and wax mulch soil treatments on tomato fruit disease control. Plant Dis.
Rep. 47:111-113.

 

 

 

 

43

Cuppels, D.C., R.A. Moore, and V.L. Morris. 1990. Construction and use of a
nonradioactive DNA hybridization probe for detection of Pseudomonas syringae pv.
tomato on tomato plants. Appl. Env. Microbiol. 56:1743-1749.

de Bruijn, F.J. 1992. Use of repetitive (repetitive extragenic palindromic and
enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain
reaction to ﬁngerprint the genomes of Rhizobium meliloti isolates and other soil
bacteria. Appl. Eviron. Microbiol. 58:2180-2187.

Denny, T.P., M.N. Gilmour, and R.K. Selander. 1988. Genetic diversity and
relationships of two pathovars of Pseudomonas syringae. J. Gen. Microbiol.
34:1949-1960.

de Vries, R. 1990. Variation among ﬁrst generation somaclonal and irradiated tomato
progeny in response to Clavibacter michiganensis subsp. michiganensis. M.Sc. 128 pp.

Dhanvantari, ED. 1989. Effect of seed extraction methods and seed treatments on
control of tomato bacterial canker. Can J. Plant Pathol. 11:400-408.

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

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

Dillard, HR. 1992. Colletotrichum coccodes: The pathogen and its hosts. p 225-236.
In: J.A. Bailey and M.J. Jerger (eds.). Colletotrichum: Biology, Pathology and Control.
Br. Soc. Plant Pathol. CAB International.

Dillard, HR. 1990. Survival of Colletotrichum coccodes in New York Phytopathology
80: 1026(abstr).

Doidge, EM. 1921. A tomato canker. Ann. Appl. Biol. 7:407-430.

Doss, B.D., J.L. Tumer, and CE. Evans. 1981. Inﬂuence of tillage, nitrogen, and rye
cover crop on growth and yield of tomatoes. J. Amer. Soc. Hort. Sci. 106:95-97.

Dowson, W.J. 1949. Manual of bacterial plant diseases. Adam & Charles Black,
London. 183 pp.

Drost, D. 1983. Response of ﬂuid sown and transplanted tomato in reduced tillage
systems. MS Thesis. Michigan State University. p. 123.

 

 

 

44

Dye, D.W., M.P. Starr, and H. Stolp. 1964. Taxonomic classiﬁcation of Xanthomonas
vesicatoria based upon host speciﬁcity, bacteriophage sensitivity and cultural
characteristics. Phytopathol. Z. 51:394-407.

Ellis, J.B., and GB. Martin. 1882. Macrosporium solani E&M. Amer. Naturalist
1611003.

Ellis, M.B., and I.A.S. Gibson. 1975. Altemaria solani. CMI Descriptions of
Pathogenic Bacteria and Fungi No. 475.

Esquivel, EA. 1984. Pleospora solani sp. nov. teleomorphisis de Altemaria solani (Ell.
& Mart.) Jones & Grout. Phytopathology 74:1014 (abstr).

FAO UN. 1984. Analysis and design of integrated crop management programs.
Working paper No. AGP:Pest/84/WP/3:4. FAO Committee of Experts on Pest Control.

Farley, JD. 1972. A selective medium for assay of Colletotrichum coccodes in soil.
Phytopathology 62: 1288-1293.

Farmer, J .V. 1959. Infection studies on tomato anthracnose caused by Colletotrichum
atmmentarium (B & BR) Taub. MS Thesis, The Univ. of Western Ontario.

Ferrandino, F.J., and W.H. Elmer. 1992. Reduction in tomato yield due to Septoria leaf
spot. Plant Dis. 76:208-211.

Fischer, S.L. 1986. The inﬂuence Of chlorothalonil, nitrogen, and straw mulch on early
blight development and yield of tomatoes. MS. Thesis, Penn. State Univ. pp. 48.

Flood, J ., and J. Rees. 1986. Host produced toxins associated with antagonism by
Aureobasidium pullulans against Altemaria solani on wounded tomato leaves. Physiol.
and Molec. Plant Pathol. 28:79—88.

Francis, C.A., D. Sander, and A. Martin. 1987. Search for a sustainable agriculture.
Crops & Soils. Aug-Sepzl2-14.

Fretz, T.A., D.R. Keeney, and SB. Sterrett. 1993. Sustainability: Deﬁning the new
paradigm. HortTechnology 3:118—126.

Frye, W.W., and R.L. Blevins. 1989. Economically sustainable crop production with
legume cover crops and conservation tillage. J. Soil Water Conserv. 44:57 -60.

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

Gabriel, D.W. 1989. The genetics of plant pathogen structure and host-parasite
speciﬁcity. Pages 343-379 in: Plant-Microbe Interaction 111. T. Kosuge and E.W.
Nester, eds. Macmillan Publishing Co., New York.

 

 

 

 

45

Gardner, RG. 1988. NC EBR-l and NC EBR-2 early blight resistant tomato breeding
lines. HortScience 23:779-780

Gardner, M.W., and J.B. Kendrick. 1921. Bacterial spot of tomato. J. Agric. Res.
21:123-156.

Getz, 8., CT Stephens, and D.W. Fulbright. 1983. Inﬂuence of developmental stage on
susceptibility of tomato fruit to Pseudomonas syringae pv. tomato. Phytopathology
73:36-38

Getz, S., D.W. Fulbright, and CT. Stephens. 1983. Scanning electron microscopy of
infection sites and lesion development on tomato fruit infected with Pseudomonas
syringae pv. tomato. Phytopathology 73:39-43.

Gilson, E., J.M. Clement, D. Brutlag, and M. Hofnung. 1984. A family of dispersed
repetitive extragenic palindromic DNA sequences in E. coli. The EMBO J.
3:1417-1421.

Gitaitis, R.D., M.J. Sasser, R.W. Beaver, T.B. McIrmes, and RE. Stall. 1987. Pectolytic
xanthomonads in mixed infections with Pseudomonas syringae pv. syringae, P.

syringae pv. tomato, and Xanthomonas campestris pv. vesicatoria. Phytopathology
77:61 1-615.

Gitaitis, RD, and R.W. Beaver. 1991. Detection of Clavibacter michiganensis subsp.
michiganensis in symptomless tomato transplants. Plant Disease 75:834-838.

Gitaitis, R.D., S.M. McCarter, and J.B. Jones. 1992. Disease contol in tomato
transplants produced in Georgia and Florida. Plant Dis. 76:651-656.

Gleason, M.L., R.D. Gitaitis, and MD. Ricker. 1993. Recent progress in understanding
and controlling bacterial canker of tomato in eastern North America. Plant Dis.
77:1069-1076.

Gleason, M.L., B]. Braun, W.M. Carlton, and RH. Peterson. 1991. Survival and
dissemination of Clavibacter michiganensis subsp. michiganensis in tomatoes.
Phytopathology 81:1519-1523.

Gonzalez, LC, and LB. Owen. 1963. Soil rot of tomato caused by Rhizoctonia solani.
Phytopathology 53:82-85.

Goode, M.J., and M. Sasser. 1980. Prevention— The key to controlling bacterial spot
and bacterial speck of tomato. Plant Dis. 64:831-834.

Grajauskis, JJ. 1990. Effects of nitrogen, rye cover and zone tillage on the yield of

 

 

fresh market tomatoes in three tillage systems. MS Thesis. Michigan State University.

70 pp.

 

 

46

Harrison, M.D., C.H. Livingston, and N. Oshima. 1965. Control of potato early blight
in Colorado. I. Fungicidal spray schedules in relation to the epidemiology of the
disease. Amer. Potato J. 42:319-327.

Hartung, IS and EL. Civerolo. 1987. Genomic ﬁngerprints of Xanthomonas
campestris pv. citri strains from Asia, South America, and Florida. Phytopathology
77:282-285.

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

Heath, M.C. 1991. The role of gene-for—gene interactions in the determination of host
species Speciﬁcity. Phytopathology 81:127-130.

Hedgwood, C.P.,Jr., G.R. Tupper, and EL. Moore. 1978. Minimum tillage for
tomatoes. Amer. Veg. Grower 16:16,18.

Henning, R.G., and LI Alexander. 1959. Evidence of existence of physiologic races of
Altemaria solani. Plant Dis. Rep. 43:298-308.

Hiemstra, H.D., and J.W. Bauder. 1984. Conservation tillage: Strategies for the future.
Proc. Conf. Conserv. Tillage. Conserv. Tillage Information Center. Fort Wayne, IN,
100 pp.

Higgins, GE, GE Ames, W.M. Barnes, J.M. Clement, and M. Hofnung. 1982. A
novel intercistronic regulatory element of prokaryotic operons. Nature 298:760-762.

Horsfall, J .G., and J .W. Heuberger. 1942. Causes, effects and control of defoliation on
tomatoes. Conn. Agr. Exp. St Bull. 456.

Hulton, C.S.J., C.F. Higgins, and RM. Sharp. 1991. ERIC sequences: a novel family of
repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and
other enterobacteria. Mol. Microbiol. 5:825-834.

 

lllrnan, W.I., R.A. Ludwig, and J. Farmer. 1959. Anthracnose of canning tomatoes in
Ontario. Can J. Bot 37:1237—1246.

Jardine, D.J., C.T. Stephens, and D.W. Fulbright. 1988. Potential sources of initial
inoculum for bacterial speck in early planted tomato crops in Michiganzdebris and
volunteers from previous crops. Plant Dis. 72:246-249.

J ardine, D.J., and CT. Stephens. 1987. Inﬂuence of timing of application and chemical
on control of bacterial speck of tomato. Plant Dis. 72:405-408.

 

 

 

47

Jensen, M.A., J.A. Webster, and N. Straus. 1993. Rapid identiﬁcation of bacteria on the
basis of polymerase chain reaction—ampliﬁed ribosomal DNA spacer polymorphisms.
Appl. Environ. Microbiol. 59:945—952.

Johnson, D.A., and ER. Miliczky. 1993. Effects of wounding and wetting duration on
infection of potato foliage by Colletotrichum coccodes. Plant Dis. 77 :13-17.

Jones, JP, and J.B. Jones. 1986. Control of early blight of tomato with foliar spray
mixtures and high fertilizer rates. Proc. Fla. State Hort. Soc. 99:

Jones, C.W., and SM. McCarter. 1974. Etiology of tomato fruit rots and evaluation of
cultural and chemical treatments for their control. Phytopathology 64: 1204-1208.

Jones, J.B., J.P. Jones, R.E. Stall, and TA. Zitter. 1991. Compendium of Tomato
Diseases. APS Press, St. Paul, MN. 73 pp.

Jones, J.B., G.V. Minsavage, RE. Stall, R.O. Kelly, and H. Bouzar. 1993. Genetic
analysis of a DNA region involved in the expression of two epitopes associated with
lipopolysaccharide in Xanthomonas campestris pv. vesicatoria. Phytopathology
83:551-556.

Jong J. and S. Honma. 1976. Inheritance of resistance to Clavibacter michiganense in
the tomato. J. Hered. 14:79—84.

Judd, A.K., M. Schneider, M.J. Sadowsky, and F.J. de Bruijn. 1993. Use of repetitive
sequences and the polymerase chain reaction technique to classify genetically related
Bradyrhizobium japonicum serocluster 123 strains. Appl. Environ. Microbiol.
59:1702-1708.

Kearney, R, P.C. Ronald, D. Dahlbeck, and B.J. Staskawicz. 1988. Molecular basis for
evasion of plant host defence in bacterial spot disease of pepper. Nature 332:541-543.

Kelly, WC. 1990. Minimum use of synthetic fertilizers in vegetable production.
HortScience 25:168—169.

Kenimer, A.L., S. Mostaghimi, R.W. Young, T.A. Dillaha, and V.O. Shanholtz. 1986.
Effects of residue cover on pesticide losses from conventional and no tillage systems.
Amer. Soc. Agr. Eng. Paper No. 86-2541, p.1-11.

Knavel, D.E., J. Ellis, and J. Morrison 1977. The effects of tillage systems on the
performance and elemental absorption by selected vegetable crops. J. Amer. Soc. Hort.
Sci. 102:323-327.

Kobayashi, D. Y., S]. Tamaki, and NT. Keen. 1989. Cloned avirulence gene from
tomato pathogen Pseudomonas syringae pv. tomato confer cultivar speciﬁciﬁcity on
soybean. Proc. Natl. Acad. Sci. USA 86:157-161.

 

 

 

 

48

Koeuth, T., J. Versalovic, and JR. Lupski. 1993. Differential subsequence conservation
supports the mosaic nature of interspersed repetitive BOX elements in bacteria.
Submitted.

Komm, D.A., and W.R. Stevenson. 1978. Tuber-bome infection of Solanum tuberosum
'Superior’ by Colletotrichum coccodes. Plant Dis. Rep. 62:682-687.

Krawiec, S. 1985. Minireview: Concept of a bacterial species. Int. J. Syst. Bacteriol.
35:217-220.

Lawson, V.F. and W.L. Surmners. 1984. Disease reaction of diverse sources of
Lycopersicon to Xanthomonas campestris pv. vesicatoria pepper strain race 2. Plant
Dis. 14:117-119.

Lawson, V.F. and W.L. Summers. 1984. Resistance to Pseudomonas syringae pv.
tomato in wild Lycopersicon species. Plant Dis. 14:139-141.

Lawton, M.B., and B.H. MacNeill. 1986. Occurrence of race 1 of Pseudomonas
syringae pv. tomato on ﬁeld tomato in southwestern Ontario. Can. J. Plant Pathol.
8:85-88.

Leach, J.B., M.L. Rhoads, C.M. Vera Cruz, F.F. White, T.W. Mew, and H Leung.
1992. Assessment of genetic diversity and population structure of Xanthomonas oryzae
pv. oryzae with a repetitive DNA element. Appl. Environ. Microbiol. 58:2188-2195.

Leben and Daft. 1965. Biological control of A.Solani and C.coccodes on tomato.

Levy, M., J. Romao, M.A. Marchetti, and J.B. Hamer. 1991. DNA ﬁngerprinting with
dispersed repeated sequences resolves pathotype diversity in the rice blast fungus. The
Plant Cell 3:95-102.

Lewis, J.A., T.H. Barksdale, and G.C. Papavizas. 1990. Greenhouse and ﬁeld studies
on the biological control of tomato fruit rot caused by Rhizoctonia solani. Crop
Protection 9:8-14.

 

Lewis, J .A., and G.C. Papavizas. 1970. Evolution of volatile sulphur-containing
compounds from decomposition of crucifers in Soil. Soil Biol. Biochem. 2:239-246.

Lupski, J .R. and GM. Weinstock. 1992. Short, interspersed repetitive DNA sequences
in prokaryotic genomes. J. Bacteriol. 174:4525-4529.

Mackenzie, DR. 1981. Association of potato early blight, nitrogen fertilizer rate, and
potato yield. Plant Dis. 65:575-577.

MacNab, AA. 1980. Tomato bacterial speck and early blight control with fungicides.
Fungic. Nematic. Tests 36:161.

 

 

 

 

49

Madden, L.V., S.P. Pennypacker, A.A. MacNab. 1978. FAST, a forecast system for
Altemaria solani on tomato. Phytopathology 68:1354-1358

Maeiro, M., T.J. N g, T.H. Barksdale. 1990. Genetic resistance to early blight in tomato
breeding lines. HortScience 25:344-346

Maiero, M., T.J. Ng, and TH. Barksdale. 1989. Combining ability estimates for early
blight resistance in tomato. J. Amer. Soc. Hort. Sci. 114:118-121

Marco, G.M., and RE. Stall. 1983. Control of bacterial spot of pepper intiated by
strains of Xanthomona campestris pv. vesicatoria that differ in sensitivity to copper.
Plant Dis. Rep. 67:779-781.

Martin, W.H. 1920. Studies on tomato leaf spot control. New Jersey Agr. Exp. Sta.
Bull. 345.

Martin, B., O. Humbert, M. Camara, E. Guenzi, J. Walker, T. Mitchell, P. Andrew, M.
Prudhomme, G. Alloing, R. Hakenbeck, D.A. Morrison, G.J. Boulnois and J.-P.
Claverys. 1992. A highly conserved repeated DNA element located in the chromosome
of Streptococcus pneumoniae. Nucleic Acids Res. 20:3479-3483.

 

McCarter, S.M., and TH. Barksdale. 1977. Effectiveness of selected chemicals in
controlling Rhizoctonia fruit rot of tomato in greenhouse and ﬁeld tests. Plant Dis. Rep.
61:811-815.

 

McCarter, S.M., J.B. Jones, R.D. Gitaitis, and DR. Smitley. 1983. Survival of
Pseudomonas syringae pv. tomato in association with tomato seed, soil, host tissue, and
epiphytic weed hosts in Georgia. Phytopathology 73:1393-1398.

McInnes, T.M., R.G. Gitaitis, S.M. McCarter, C.A. Jawarski, and SC. Phatak. 198 .
Airborne dispersal of bacteria in tomato and pepper transplant ﬁelds Plant Dis.
72:575—579. -

McKeown, A.W., R.F. Cerkauskas, and J .W. Potter. 1988. Inﬂuence of strip tillage on
yield, diseases and nematodes of tomatoes. J. Amer. Soc. Hort. Sci. 113:328-331.

Merwin, LA, and MP. Pritts. 1993. Are modern fruit production systems sustainable'l.
HortTechnology 3:128-136.

Miller, A.N., T.J. NG, and T. H. Barksdale. 1984. Comparison of inheritance of
resistance to tomato anthracnose caused by two Colletotrichum spp. Plant Dis.
68:87 5—877.

Minsavage, G.V., D. Dahlbeck, M.C. Whalen, B. Kearney, U. Bonas, B.J. Stakawicz
and RE. Stall. 1990. Gene-for-gene relationships specifying disease resistance in
Xanthomonas campestris pv. vesicatoria-pepper interactions. Mol. Plant-Microbe
Interact. 3:41-47.

 

 

50

Mojtahedi, H., G.S. Santo, J.H. Wilson, and AN. Hang. 1993.
Managing Meloidogyne chitwoodi on potato with rapeseed as green manure. Plant Dis.
77:42—46.

Moore, W.D. 1942. Some factors affecting the infection of tomato seedlings by
Altemaria solani. Phytopathology 32:399-403.

Moore, W.D., H.R. Thomas, and BK. Vaughn. 1943. Tomato seed treatment in relation
to control of Altemaria solani. Phytopathology 33:797-805.

Morse, RD. 1993. Components of sustainable production systems for vegetables-
conserving soil moisture. HortTechnology 3:211-214.

Muehlchen, A.M., R.E. Rand, and J.L. Parke. 1990. Evaluation of crucifer green
manures for controlling Aphanomyces root rot of peas. Plant Dis. 74:651-654.

Nash, A.F., and R.G. Gardner. 1988. Heritability of tomato early blight resistance
derived from Lycopersicon hirsutum P.I. 126445. J. Amer. Soc. Hort. Sci. 113:264-268

Nash, A.F., and R.G. Gardner. 1988. Tomato early blight resistance in a breeding line
derived from Lycopersicon hirsutum PI 126445. Plant Dis. 72:206-209

O’Leary, DO. 1985. Effects of fungicides and host resistance on epidemics of tomato
earl blight. Ph.D. Thesis. North Carolina State University. 17 5 pp.

OMAF. 1991. Agricultural statistics. Ontario Ministry of Agriculture and Food. Queens
Park, Toronto.

Pantidou, M.B., and WT. Schroeder. 1955. Foliage as a secondary source Of inoculum
for tomato anthracnose. Phytopathology 45 :338-345.

Papavizas, G.C., P.B. Adams, RD. Lurnsden, J.A. Lewis, R.L. Dow, W.A. Ayers, and
J.G. Kantes. 1975. Ecology and epidemiology of Rhizoctonia solani in ﬁeld soils.
Phytopathology 65:871-877.

Parmeter, J.R.,Jr., (ed.). 1970. Rhizoctonia solani, Biology and Pathology. Univ. of
California Press. 255 pp.

Patterson, CR 1991. Importance of chlamydospores as primary inoculum for
Altemaria solani incitant of collar rot and early blight on tomato. Plant Disease 75:

Pennypacker, S.P., L.V. Madden, and AA. MacNab. 1983. Validation of an early
blight forecasting system for tomatoes. Plant Dis. 7:287-289.

Peterson, GP. 1963. Survival of Xanthomonas vesicatoria in soil and diseased tomato
plant. Phytopathology 53:765-767.

 

 

 

 

51

Petrunak, D.M., and B.J. Christ. 1992. Isozyme variability in Altemaria solani and A.
alternata. Phytopathology 82:1343-1347.

Phillips, R.E., R.L. Blevins, G.W. Thomas, W.W. Frye, and SH. Phillips. 1980. NO-till
agriculture. Science 208: 1 108-1 1 13.

Pilowsky, M., and O. Zutra. 1982. Screening wild tomatoes for resistance to bacterial
speck pathogen (Pseudomonas tomato). Plant Dis. 66:46-47.

Pitblado, RP. 1988. Developement of a weather-timed fungicide spray program for
ﬁeld tomatoes. Can. J. Plant Path. 10:371 (abstr).

Pitblado, RE. 1992. Development and implementation of Tom-Cast. Ont. Min. A gr.
and Food. Publ. p.18.

Pitblado, RE, and EA. Kerr. 197 9. A source of resistance to bacterial speck -
Pseudomonas tomato. Tomato Genet. Coop. Rpt. 9:30.

Pohronezny, K., and RB. Volin. 1983. The effect of bacterial spot on yield of fresh
market tomatoes. HortScience 18:69-70.

Poincelot, RP. 1986. Toward a more sustainable agriculture. AVI, Westport, Conn.

Pound, GS. 1951. Effect of air temperature on incidence and development of the early
blight disease of tomato. Phytopathology 41:127-135.

Precheur, R.P., R.R. Riedel, M.B. Bennett, K.L. Wiese, and Dudek, J. 1992.
Management of fungicide residues on processing tomatoes. Plant Disease 76:700-702.

Price, HQ, and RA. Baughan. 1987 . Establishment of fresh market tomatoes in a no-
till system. Acta Hort. 198:261-268.

Pritchard, F.J., and W.S. Porte. 1921. Collar rot of tomato. J. Agric. Res. 21:179-184.
Pscheidt, J .P., and W.S. Stevenson. 1986. Early blight of potato and tomato: A
literature review. College of Agr. and Life Sci. Res. Rep. NO. 3376. Univ. of

Wisconsin—Madison. 17p.

Putnam, AR. 1990. Vegetable weed control with minimum herbicide inputs.
HortSicence 25:155-159.

Raid, RN, and SP. Pennypacker. 1987. Weeds as hosts for Colletotrichum coccodes.
Plant Dis. 71:643-646.

Rands, RD. 1917. Early blight of potato and related plants.
Wis. Agric. Exp. Stn. Res. Bull. 42, 48 pp.

 

 

 

52

Robinson, RA. 1976. Plant Pathosystems. Springer-Verlag, Berlin, Heidelberg, NY,
184 pp.

Rotem, J ., B. Wooding, and DE. Aylor. 1985. The role of solar radiation, especially
ultraviolet, in the mortality of fungal spores. Phytopathology 75:510-514

Rotem, J. 1969. The effect of soil moisture level on the incidence of early blight on
potato and tomato plants. Isr. J. Agric. Res. 19:139-141.

Rotem, J., and L. Reichert. 1964. Dew - a principle moisture factor enabling early
blight epidemics in a semiarid region of Israel. Plant Dis. Rep. 48:211-215.

Rotem, J., and E. Bashi. 1969. Induction of sporulation of Altemaria f.sp. solani by
inhibition of its vegetative development. Trans. Br. Mycol. Soc. 53:433-439.

Rotem, J. 1964. The effect of weather on the dispersal of Altemaria spores in a semi
arid region of Israel. Phytopathology 54:628-632.

Rotem, J. 1965. Sand and dust storms as factors leading to Altemaria blight epidemic
on tomatoes and potatoes. Agr. Meteorology 2:281-288.

Rush, CM. and SR. Winter. 1990. Inﬂuence of previous crop on Rhizoctonia root and
crown rot of sugar beet. Plant Dis. 74:421-425.

Sarrantonio, M. 1992. Opportunities and challenges for the inclusion of soil-improving
crop in vegetable production systems. HortScience 27:754-758.

Scott, J.W., and J.B. Jones. 1986. Sources of resistance to bacterial spot in tomato.
HortScience 21:304-306.

Selander, R.K. and J .M. Musser. 1990. Population genetics of bacterial pathogenesis, p.
11-36. In B.H. Iglewski and V.L. Clark (ed.), Molecular basis of bacterial pathogenesis.
Academic Press, Inc., San Diego, Calif.

Sharples, OJ. and R.G. Lloyd. 1990. A novel repeated DNA sequence located in the
intergenic regions of bacterial chromosomes. Nucl. Acids Res. 18:6503-6508.

Shelby, P.P.,Jr., D. L. Coffey, N.,Jr. Rhodes, and L. S. Jeffery. 1988. Tomato
production and weed control in no-tillage versus conventional tillage. J. Amer. Soc.
Hort. Sci. 113:675-678.

Sherman, C. 1992. Cover crops, nitrogen cycling, and soil properties in semi-irrigated
vegetable production systems. HortScience 27:749-754.

Sherf, A.F., and AA. MacN ab. 1986. Vegetable diseases and their control. 2nd edition,
John Wiley & Sons, NY. 728 pp.

 

 

 

 

53

Shoemaker, RB. 1976. Fungicide evaluations for tomato early blight. Fungic. Nematic.
Tests 31:108-109.

Shoemaker, RB. 1980. Fungicides, resistance and spray timing for tomato early blight.
Fungic. Nematic. Tests 35:95-96.

Sijam, K., C]. Chang, and RD. Gitaitis. 1991. An agar medium for the isolation and
identiﬁcation of Xanthomonas campestris pv. vesicatoria from seed. Phytopathology
81:831-834.

Smith, ER 1910. A new tomato disease of economic importance. Science 31:794-796.

Spieser, H. 1983. Feasibility of strip-cultivation in processing tomatoes. Ont. Min. A gr.
and Food. Agdex 230/741.

Stall, RE, and PL. Thayer. 1962. Streptomycin resistance of the bacterial spot
pathogen and control with streptomycin. Plant Dis. Rep. 46:389—392.

Stall, R.E., C. Beaulieu, D. Egel, N.C. Hodge, R.P. Leite, G.V. Minsavage, H. Bouzar,
J.B. Jones, A.M. Alvarez, and AA. Benedict. 1994. Two genetically diverse groups of
strains are included in Xanthomonas campesm's pv. vesicatoria. Int. J. System.
Bacteriol. 44:47-53.

Stephens, C.T. 1990. Minimizing pesticide use in a vegetable management system.
HortScience 25:164-168.

Stern, V.M., R.F. Smith, R. van den Bosch, and KS. Hagan. 1959. The integrated
control concept. Hilgardia 29:81-101.

Stevenson, RE, and SP. Pennypacker. 1988. Effect of radiation, temperature, and
moisture on conidia] germination of Altemaria solani. Phytopathology 7 8:926-930.

Stevenson, W.R. 1977. Use of captafol and chlorothalonil on reduced application
method schedules for tomato disease control in Indiana.Plant Dis. Rep. 61:803-805.

Stevenson, W.R., G.E. Evans, and TH. Barksdale. 1978. Evaluation of tomato breeding
lines for resistance to fruit anthracnose. Plant Dis. Rep. 62:937-940.

Strider, BL. 1969. Bacterial Canker of tomato caused by Corynebacten'um
michiganense. Tech. Bull. 193. North Carolina Agric. Exp. Stn. 110 pp. ..

Sumner, DR, 8., Jr. Doupnik, and MG. Boosalis. 1981. Effects of reduced tillage and
multiple cropping on plant diseases. Annu. Rev. Phytopathol. 19:167-187.

Sumner, D.R., et al. 1986. Interactions of tillage and soil fertility with root diseases in
snap bean and lima bean in irrigated multiple cropping systems. Plant Dis. 70:730-735.

 

 

 

 

 

54

Sumner, D.R., E.D. Threadgill, D.A. Smittle, S.C. Phatak, and AW. Johnson. 1986.
Conservation tillage and vegetable diseases. Plant Dis. 70:906-911.

Swarup, S., R. De Feytr, and D.W. Gabriel. 1991. A pathogenicity locus from
Xanthomonas citri enables strains from several pathovars of X. campestris to form
cankerlike lesions on citrus. Phytopathology 81:802-809.

Swarup, S., Y. Yang, M.T. Kinsley and D.W. Gabriel. 1992. An Xanthomonas citri
pathogenicity gene, pthA, pleiotrpically encodes gratuitous avirulence on nonhosts.
Mol. Plant-Microbe Interact. 3:204-213.

Thomas, HR. 1944. ”Freckle,” a spotting of tomato fruits. Phytopathology 34:341-344.

Thomas, HR. 1948. Effect of nitrogen, phosporous, and potassium on susceptibility of
tomatoes to Altemaria solani. J. Agric. Res. 76:289-306.

Thompson, B.T., J.L. Leary, and WC. Chun. 1989. Speciﬁc detection of Clavibacter
michiganense subsp. michiganense by a homologous DNA probe. Phytopathology
14:31 1-314.

Thyr, B.T. 1968. Resistance to bacterial canker in tomato, and its evaluation.
Phytopathology 58:279-281.

USDA 1990. Agriculture statistics. United States Department of Agr. US. Government
Printing Ofﬁce. Washington, DC.

Vakili, NS. 1967. Importance of wounds in bacterial spot (Xanthomonas vesicatoria) of
tomatoes in the ﬁeld. Phytopathology 57: 1099-1103.

van Bruggen, A.H.C., P.R. Brown, C. Sherman, and AS. Greathead. 1990. The effect
of cover crops and fertilization with ammonium nitrate on corky root of lettuce. Plant
Dis. 74:584-589.

Vauterin, L., J. Swings, K. Kersters, M. Gilles, T.W. Mew, M.N. Schroth, NJ.
Palleroni, D.C. Hildebrand, D.E. Stead, E.L. Civerolo, A.C. Hayward, H. Maraite, RE.
Stall, A.K. Vidaver, and J .F. Bradbury. 1990. Toward an improved taxonomy of
Xanthomonas. Int. J. Syst. Bacteriol. 40:312-316.

Versalovic, J., T. Koeuth, and J .R. Lupski. 1991. Distribution of repetitive DNA
sequences in eubacteria and application to ﬁngerprinting of bacterial genomes. Nucleic
Acids Res. 19:6823-6831.

Volcani, Z. 1969. The effect of mode of irrigation and wind direction on disease
severity caused by Xanthomonas vesicatoria. Plant Dis. Rep. 53:459-461.

Waggoner, PE, and JG. Horsfall. 1969. EPIDEM- A simulator for plant disease
written for a computer. Conn. Agr. Exp. St. Bull. 698. 80 pp.

 

 

 

55

Wallace, R.W., and RR. Bellinder. 1992. Alternative tillage and herbicide options for
successful weed control in vegetables. HortScience 27:745-749.

Waney, V.R., M.T. Kingsley, and D.W. Gabriel. 1991. Xanthomonas campestris pv.
translucens genes determining host—speciﬁc virulence and general virulence on cereals
identiﬁed by TnS-gUSA insertion mutagenesis. Mol. Plant-Microbe Interact. 4:623-627.

Wang, J.F., J.B. Jones, J.W. Scott, and RE. Stall. 1990. A new race of the tomato
group of strains of Xanthomonas campestris pv. vesicatoria. Phytopathology 8021070
(abstr).

Weirr, HQ 1990. Sustainable commercial vegetable production with minimal use Of
synthetic fertilizers and pesticides: a postlude. HortScience 25: 170—171.

Wellman, FL. 1943. A technique to compare virulence of Altemaria solani on tomato
leaﬂets. Phytopathology 33:698-706.

Welsh, J. and M. Mcclelland. 1990. Fingerprinting genomes using PCR with arbitrary
primers. Nucleic Acids Res. 18:7213-7218.

Whalen, M.C., R.E. Stall, and B.J. Staskawicz. 1988. Characterization of a gene from a
tomato pathogen determining hypersensitive resistance in non-host species and genetic
analysis of this resistance in bean. Proc. Natl. Acad. sci. USA 85:6743-6747.

Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990.
DNA polymorphisms ampliﬁed by arbitrary primers are useful as genetic markers.
Nucleic Acids Res. 18:6531-6535.

Wilson, ID, and HA. Runnels. 1949. Comparitive susceptibility of tomato varieties to
anthracnose and their response to spraying. Ohio Agr. Exp. St. Bull. 685.

Woods, C.R., J. Versalovic, T. Koeuth, and J .R. Lupski. 1992. Analysis of relationships
among isolates of Citrobacter diversus by using DNA ﬁngerprints generated by

repetitive sequence-based primers in the polymerase chain reaction. J. Clin. Microbiol.
30:2921-2929.

Younkin, S.G., N.J. Riverton, and A.W. Dimock. 1944. Foliage infection of
Lycopersicum esculentum by Colletotrichum phomoides. Phytopathology 34:976-977.

Yunis, H., Y. Bashan, Y. Okon, and Y. Herris. 1980. Weather dependence, yield losses,
and control of bacterial speck of tomato caused by Pseudomonas tomato. Plant Dis.
64:937-939.

 

 

 

 

CHAPTER II

INTEGRATED MANAGEMENT OF EARLY BLIGHT, ANTHRACNOSE AND
SOIL ROT OF TOMATO WITH REDUCED FUNGICIDE USAGE

AND CULTURAL PRACTICES

56

 

 

 

 

57

ABSTRACT

The integration of a reduced-sprays program and conservation tillage was
studied in 1990 - 1992 in fresh market (FMT) and processing tomato (PRT)
(Lycopersicon esculentum) production systems for the control of early blight (EB),
caused by Altemaria solani, anthracnose (ANTH), caused by Colletotrichum coccodes,
and soil rot (SR), caused by Rhizoctonia solani. Reduced-sprays were scheduled
according to TOMCAST, a program that calculates a daily disease severity value
(DSV) based on the average temperature during hours when tomato foliage is wet.
Select forecast generated chlorothalonil spray schedules required 45-80% fewer
applications but did not compromise incidence of percent PRT fruit with mold in
1990-1992 nor incidence of percent FMT fruit with mold in 1991-1992 as compared to
a standard weekly spray program. Reduced-sprays did not adequately control a high
incidence of SR on FMT fruit in 1990. Reduced sprays were most effective in disease
control when integrated with a zone tillage (ZT) system in 1990, a conventional tillage
(CT) system in the absence of rotation in 1991, and ZT or rotation in 1992. Zone
tillage, as compared to CT, reduced mean area under the disease progress curve
(AUDPC) due to BB in 1990 and in the absence of rotation in 1991, resulted in
increased AUDPC values. In 1992, ZT decreased mean AUDPC values in plots
planted to continuous tomato (no rotation) and increased mean AUDPC in tomato
plots rotated to cucumber the preceding year. Integrated disease management of EB,
ANTH and SR was possible with reduced fungicide input and cultural practices

affording advantages associated with sustained productivity of farmland.

 

 

 

 

 

58
INTRODUCTION

Early blight, caused by Altemaria solani (Ellis & Martin) Jones & Grout,
anthracnose (ANTH), caused by Colletotrichum coccodes (Wallr.) Hughes, and soil rot
(SR), caused primarily by Rhizoctonia solani Kuehn (Batson 1973; Jones & McCarter
1974) are the most important fungal diseases of tomato foliage and fruit in the
northcentral (NC) production region (MI, OH, 1N, Ontario) of North America. High
humidity and warm temperatures combined with extensive dew periods and abundant
rainfall favor EB, ANTH and SR each season. Each disease can incite losses of 35 to
70 % or more (Batson 1973; Jones & McCarter 1974; Sherf and McNab 1986; Wilson
and Runnels 1949). Therefore growers currently follow standard recommendations to
initiate fungicide sprays when fruit ﬁrst set and to apply subsequent applications every
7 to 14 days on nearly 100% of the tomato hectarage. Accordingly, twelve or more
seasonal sprays are applied with limited consideration of disease level, weather
patterns or cropping systems.

The recent removal of several ftmgicide products has currently put the tomato
industry in the tenuous position of relying, almost exclusively, on only two families of
fungicides, the ethylene-bis-dithiocarbamates (EBDCs) and chlorothalonil, for fungal
disease control. Loss of either fungicide would impinge on the ability of the
northcentral tomato region to reliably produce a marketable crop within the context of
current production systems. In addition, fungicide usage is facing an unprecedented
challenge including consumer, regulatory agency, environmental, grower-safety, and
cost of application concerns. The loss of registered materials, residue concerns, and

lack of forthcoming new chemistry requires prudent use of EBDCs and chlorothalonil

 

 

 

 

 

59

to minimize food safety concerns and ensure their long-term use until alternative
disease control strategies can be devised and implemented.

The challenge tomato growers currently face is not limited to their desire to
limit fungicide use. Conventional production systems with intense tillage of the land
and energy—intensive inputs contribute to high variable production costs and are seen
as counter-productive to a sustainable agricultural system (Fretz et al. 1993). At the
farm enterprise and ﬁeld level, components of sustainability include reduced tillage
practices, maintenance of a surface crop residue, use of cover and green manure crops,
and crop rotation (Fretz et a1. 1993; Frye and Blevins 1989; Sarrantonio 1992;
Sherman 1992). Sustainable farming practices have not been adopted with equal
success in vegetable (Kelly 1990) as compared to ﬁeld crop production systems
(Phillips et a1. 1980). In part, moldboard plowing and intensive tillage have been
crucial cultural practices for breaking the life cycle of pests that limit vegetable
productivity (Putnum 1990; Sumner et a1. 1986). Advancements in reduced tillage and
cover crop use have recently been made in numerous vegetable production systems
(Coolman and Hoyt 1993; Morse 1993; Phatak 1992; Sarrantoniol992; Sherman 1992;
Wallace and Bellinder 1992; Wien 1990) including tomato (Abdul-Bald and Teasdale
1993; Doss et al. 1981; Knavel et al. 1977; McKeown et al. 1988; Price and Baughan
1988; Shelby et a1. 1988). Unfortunately, most studies have not included a pathology
component (Sumner et al. 1986) and cannot be recommended without a greater
appreciation of the system as a whole.

The objective of this study was to reduce fungicide usage and evaluate the
potential of integrated disease management practices for the control of early blight,

anthracnose, and soil rot within the context of a fresh market (FMT) and processing

 

 

 

 

6O

tomato (PRT) production system currently under research at Michigan State
University. We validated a TOMato disease foreCASTing model, TOMCAST
(Pitblado 1988), to determine the need for fungicide sprays and we studied the impact
of reduced tillage, cover and green manure crops, and a one year rotation as they

affect the foliar-fruit fungal disease complex.

MATERIALS AND METHODS

Location and design of ﬁeld experiments. Field experiments were initiated in the fall
of 1989 and conducted in 1990, 1991 and 1992 on a Spinks sandy loam (87.4% sand,
6.0% silt and 6.6% clay) at the Southwest Michigan Research and Extension Center
near Niles, MI. The site of the experiment had been seeded to rye and was soil
incorporated September 1989. Rye (Secalis cereale cv. Wheeler) was drilled at 168 kg
ha‘1 in the fall prior to each planting season. Inoculum was not introduced but
presumably arose from indigenous sources or as transplant and seed borne inoculum.
The experiment had a split-split plot design with four replications arranged in
randomized complete blocks. Each replication comprised two main plot parcels of land
(about 12 x 84 m) side by side and separated by approximately 9 m of permanent sod.
Each main plot was subdivided into two 12 x 42 m sub-plots and each subplot was
divided into seven 12 x 6 m sub-sub-plots for a 2x2x7 factorial design (n=112). Each
plot contained four cropped rows on 1.5 m centers. Plots planted to tomato -
(Lycopersicum esculentum) consisted of two rows of a fresh market cultivar and two

rows of a processing cultivar. All data, were taken from the inner 6 m of each plot of

the inner plot-row of each tomato type. Analyses of all data were done independently

 

 

 

 

61

for processing and fresh market tomato as two simultaneous experiments. All four
rows were planted to cucumbers (Cucumis sativas) in sequence as described below. No
disease data were collected from cucumber plots and cucumber yields are reported

elsewhere (Chapter 3).

Main plots: Crapping sequence. Main plot treatments consisted of continuous tomato
(no rotation) or tomato rotated with cucumber (Figure 1), a biennial cropping system
commonly used by many growers in southwest Michigan. Tomato was transplanted
into all plots in 1990. Tomato was tranplanted into half the plots and cucumber seeded
in the other half in 1991. Immediately after the cucumber harvest (July 25), the entire
12 x 84 m main plot was plowed and drilled to a mustard crop as a rapid source of
green manure and potential fungistatic and weed suppressive properties. All plots were
conventionally tilled the third week of September and rye was drilled. In 1992, tomato

was again transplanted into all plots.

Sub-plots: Tillage system. Two tillage systems were employed. Conventional tillage
(CT) consisted of moldboard plowing to a depth of 20-23 cm when the over wintered
rye was 15-20 cm high. Up to two additional ﬁeld passes with a disk and/or drag was
employed for ﬁeld preparation prior to planting. After each tomato harvest, plots were
conventionally tilled and rye was seeded.

Zone tillage (ZT) was used as a second tillage treatment and is deﬁned as the
fracturing of the soil directly below where the plant is to be established (Grajauskis
1990). In early spring (late March to early April) of each year, paraquat (Gramoxone)

at the rate of 1.1 kg ha”1 was applied to the over wintered rye in strips 0.46 m wide on

 

 

 

62

1.5 m (row) centers. In this manner, minimal rye biomass accumulated where tomato
or cucumber plants were to be established. The inter-row rye continued to grow and
was desiccated with paraquat when it reached a height of 1 to 1.2 m. Over time, the-
inter-row rye lodged and settled to the soil surface.

In ZT plots each spring, the Tye paratill (Tye Company, Lockney, TX) was set
to fracture the soil to an approximate 35 cm depth with minor surface disturbance and
no soil inversion. After the tomato harvest in 1990, rye was drilled directly into ZT
plots with no additional tillage. The goal with ZT was to perform no additional tillage
until the summer or fall of the second year (1991). However, soil pentrometer readings
indicated sufﬁcient soil compaction occurred over the previous year to warrant a
repeated ZT procedure in the spring of 1991. Zone tillage was performed on the exact
same row center where tomato plants once stood and tomato or cucumber were to be
planted. All plots were conventionally tilled after harvest in 1991 as described above.

Figure 1 provides a summary of the overall 3 year crop production system

employing ZT and a rotation of tomato with cucumber.

Sub-Sub plots: Fungicide treatment. Plots were not sprayed, sprayed weekly, or
sprayed at intervals according to the disease forecasting model, TOMCAST.

TOMCA ST calculated a daily Disease Severity Value (DSV) based on the average
temperature during hours when leaves were wet (Table 1) similar to the FAST model
(Madden et al. 1978). Hourly mean temperature and leaf wetness were recorded using
the Omnidata model DP223 temperature and leaf wetness recorder (Omnidata

International, Inc., Logan, UT). Sensors were calibrated each year.

 

 

 

 

 

63

FIGURE 1: Summary of the overall 3 year crop production system employing zone
tillage (ZT) and rotation of tomato with cucumber. Solid lines represent crop growth.
Dotted lines represent windows of preferred time periods for agronomic inputs. Fields
are conventionally tilled (CT) commencing year one and not subject to CT again until
after the cucumber harvest in year two. After a mustard crop, Cl‘ is used once more
and the ﬁeld is planted back to tomato. Intensive two year rotations are common with
some growers in SW Michigan.

 

 

 

 

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65

TABLE 1: Number of hours of leaf wetness at a given temperature range required for
each disease severity value (DSV).

 

 

 

 

 

 

 

 

 

 

 

Mean Disease Severity Values (DSVS)
Temperature
(C) 0 1 2 3 4
13.0 - 17.5 0-6 hr 7-15 16-20 21+
17.6 - 20.5 0-3 4—8 9-15 16-22 23+
20.6 - 25.5 0—2 3-5 6—12 13-20 21+
25.6 - 29.5 0-3 4-8 9-15 16-22 23+
Hours of Leaf Wetness

 

 

 

 

TOMCAST called for an initial spray on July 11 or earlier if DSVs reached a
threshold of 35 for tomatoes planted prior to May 23 and 45 for tomatoes planted after
May 23 (Pitblado 1992). Subsequent sprays were applied after the accumulation of every
15, 20 or 25 DSVs. The fungicide Bravo 720 (chlorothalonil) was used throughout the

study at full recommended rate (4.2 L ha") or at a reduced rate (2.8 L ha"). Originally,

Dyrene was designed to be used prior to fruit set in one treatment followed by

 

chlorothalonil sprays. However, initial applications coincided with fruit set and Dyrene
did not need to be applied. Therefore, this treatment was sprayed according to a
TOMCAST schedule (DSV 20H) in 1990 or not sprayed in 1991. Although data was

collected from these plots and used to generate analysis of variance, in most cases the

 

data is not presented since the means were no different than their corresponding duplicate
treatments. Fungicide treatments, rates, and date of ﬁrst application in each year are
outlined in Table 2. Fungicides were applied with a hand- held boom connected with high

pressure hose to an FMC tractor drawn sprayer. The boom width was adjusted with plant

 

 

66

TABLE 2: Date of harvest, date and rate of ethrel treatment, fungicide treatment, number
of fungicide applications, and date of initial fungicide application in years 1990 to 1992.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ACTIVITY OR YEAR
TREATMENT
1990 1991 1992
HARVEST DATES Aug 7(218)‘ Jul 30(210) Aug 19(231)
OF FRESH MARKET
TOMATO Aug 15(226) Aug 5(216) Aug 25(237)
Aug 22(233) Aug 12(223) Sep 1(244)
Aug 28(239) Aug 21(232) Sep 9(252)
Sep 5(247) Aug 27(238) Sep 15(258)
Sep 12(254) Sep 22(265)
HARVEST DATE Sep 18(260) Aug 29(240) Sep 29(273)
OF PROCESSING
TOMATO
DATE OF ETHREL APPLN Sep 5 Aug 16 Sep 14
RATE OF ETHREL 2.8 L ha" 4.2 L ha" 4.2 L ha"
APPLIED
FUNGICIDE TREATMENT NUMBER OF FUNGICIDE APPLICATIONS
WEEKLY 15(Jun 15)” 11 (Jun 16) 13 (Jun 25)
DSVc 15Ld NA‘ 6 (JUN 26)” 5 (Jul l6)b
st 153 NA 6 (Jun 26) 4‘
DSV 20L 4 (Jul 11)” 4 (Jun 26) 4 (Jul 16)
4 (Jul 11) 4 (Jun 26) 3f
DSV 25L 3 (Jul 11) NA NA
DSV 25H 3 (Jul 11) NA 3 (Jul 16)
NO SPRAY 0 0 0

 

 

 

 

 

 

; Julian Day of Year
Date of intitial application for weekly or TOMCAST-based spray programs

i° Fungicide applied after the accumulation of every 15, 20 or 25 disease severity values
‘ L - low rate of Bravo 720 (2.8 lira"), H - high rate of Bravo 720 (4.2 L ha")
° Treatment not applied during this year

' Initial spray was inadvertarltly omitted. First application = Jul 30

 

 

 

67
growth to a maximum of 1.2 m and had four swivel T-Jet nozzles, two at the boom and
two as 35 cm drop nozzles, to ensure adequate coverage. The pressure was 667 kPa at

the sprayer pump and a total volume of 836 L ha‘1 was applied.

Other cultural practices. Each year nitrogen (ammonium nitrate 33-0—0) was broadcast
over the rye in all plots in early April at the rate of 56 kg ha“. Cucumber and tomato
received additional N at the rate of 56 kg ha’1 pre-plant incorporated in CI‘ plots or
banded at planting in ZT plots. An additional 28 kg ha'1 was sidedressed approximately
3 weeks after ﬁeld setting tomato plants. An additional 56 kg ha'1 and 28 kg ha‘1 was
sidedressed to cucumber plots at the 3 true leaf stage and tip over, respectively.
Sidedressed N was applied as a band on the soil surface beside each row. Phosphorous
and potassium were applied according to recommended rates for cucumber or tomato
based on soil fertility tests conducted each fall.

Trifluralin (Treﬂan) at the rate of 0.56 kg ha'1 was preplant incorporated in CT
plots for control of germinating weed seeds. Post-planting weed control was achieved
with cultivation in CT plots. In ZT plots, metribuzin (Sencor) was used at the rate of
0.34 kg ha“, for postemergent control of broadleaf weeds, and Fusilade was used at
the rate of 0.3 kg ha‘l, for post-emergent control of grass weeds. Herbicide and
cultivation were complimented with hand hoeing as required. Curbit was pre-plant
incorporated at recommended rates for weed control in cucumber CT plots. Curbit
and/or Fusilade, complemented with cultivation (CT plots only), and hand hoeing was
used for postemergence weed control. Guthion was applied at recommended rates for

insect control as required.

Four to ﬁve week old commercially grown tomato seedlings in 72 (FMT) or

 

 

 

68
288 (PRT) cell ﬂats were ﬁeld set the last week of May 1990 and 1991 and ﬁrst 10
days of June 1992 using a conventional single row transplanter with double disk
openers and a wide rubber drive. The fresh market cv. 'Pik Rite’ and processing cvs.
’Ohio 7870’ (1990) and ’Heinz 8704’ (1991 & 1992) were spaced 0.6 and 0.3 m,
respectively, on 1.5 m centers and grown by conventional ground production methods
(no mulch, trellis or training). Cucumbers cv. Flurry were direct seeded early June.

Overhead sprinkler irrigation was applied as needed.

Assessment of disease incidence. Percent defoliation due to early blight (necrosis and
chlorosis) was assessed visually on a weekly basis after symptoms became apparent (1
to 2% severity in plots not sprayed) and continued until complete harvest of FMT fruit
or treatment of PRT tomato plants with Ethrel. Assessments were based on all plants
within the inner 6 In section of the inner row for each tomato type. Early season
incidence of disease in 1991 and 1992 was assessed by counting the number of lesions

per plant or percentage of plants with symptoms.

Tomato harvest and fruit mold incidence. Fresh market tomatoes were multiple
harvested (dates shown in Table 2) from a 6 m row section when fruit reached the
breaker stage or riper. All fruit were graded twice, once using market standards and
again for disease symptoms. Fruit were graded for size on a commercial grader. Sizes
included large fruit (No. 1) with a diameter >67 mm and medium (No. 1) fruit with a
diameter of 54-67 mm. Marketable fruit with blemishes were labelled No.2’s and non
marketable fruit was culled. Data collected according to market standards is presented

elsewhere (Chapter 3). Fruit were also sorted for symptoms of EB, ANTH, SR or

 

 

 

69

bacteria and disease incidence on fruit was expressed as a percentage of total fruit
weight evaluated. Data on bacterial disease incidence is presented elsewhere
(Appendix B). Processing tomatoes were treated with Ethrel and harvested by a once-
over harvest (rates and dates shown in Table 2). Fruit from the 6 m harvested area was
weighed and pooled. Subsequently, two subsamples collected in 20 L pails, were rated
by independent teams of people according to market standards for ripe, green and cull
fruit, or for incidence of ANTHR, EB or SR and expressed as a percentage of the total
weight of fruit evaluated. Samples of foliage or fruit were periodically selected and

pathogens isolated to verify causal organisms.

Data analysis. All data were tested for homogeneity of variance using Bartlett’s test
(Little and Hills 1978) before analysis of variance with Plot-IT (Scientiﬁc
Programming Enterprises, Haslett, MI) or MSTAT-C (Michigan State University, E.
Lansing, MI). Only in 1992 was a full three way factorial model used with fungicide
as a split plot of tillage and tillage as a split plot of rotation. In 1990 and 1991 a two
way factorial analysis was performed with fungicide as a split-plot of tillage using 8
(n=112) and 4 (n=56) replications, respectively.

The experiment was designed to determine the effect of rotation, tillage and
fungicide treatment and their interactions. ANOVA was used to partition the degrees
of freedom and associated sums of squares for the main factors and their associated
interactions. With no interactions, signiﬁcant effects due to rotation or tillage were
determined by planned F tests calculated from the analysis of variance table using the
appropriate error term. Means from signiﬁcant fungicide treatment effects were

separated with appropriate LSDs based on a signiﬁcant F value calculated using the

 

 

 

 

70

overall residual mean square error of the AN OVA table (i.e. protected LSD).

Certain plots were above a ﬁeld tile that malfunctioned during the experiment.
Few plots were affected in 1990. Depending on the data set, 4 to 6 of 112 plots were
outliers and new values were substituted using the MISVALEST subroutine of
MSTAT-C. One degree of freedom for each estimated value was subtracted from the
overall residual error mean square before signiﬁcance of fungicide treatment effects
was determined. Examples of substituted data are provided in the text. No plots were
affected in 1991. In 1992, the problem persisted and impacted 2 replications of a
complete treatment (i.e. numerous sub-sub-plots in CT sub-plots in rotation main
plots). With unreliable data for 2 replications of complete treatments, 1992 data were
analyzed over the remaining 2 replications only (n=56 rather than 112).

Mean areas under the disease progress curve (AUDPC) expressed as percent-

days were calculated according to the method of Shaner and Finney (1977 ):

i“ Y. + .
AUDPC=Z[———( "‘2 Y’)][(t,,l-t,-)]

where Y, = disease severity at the ith observation, t, = time (days) after the initial
rating at the ith observation, and n = total number of observations. Data were also
transformed to assess the apparent impact of initial inoculum or rate of disease
increase. The value 0.005 % was added to each observation of disease incidence prior
to transformation. The appropriateness of the logistic and Gompertz model was
determined by comparing the coefﬁcient of detennination (R2) and examination of
scatter plots of residual terms (Campbell and Madden 1990). Selected correlation. or
regression analysis among data sets were performed using Pearson’s correlation

coefﬁcient or a model that provided good ﬁt, respectively.

 

 

 

 

Mean temperature and rainfall varied considerably over the three year study
(Table 3). The ﬁrst year was relatively normal, 1991 was one of the hottest summers
on record, and 1992 was one of the coolest seasons on record with a wet Jul. High
levels of EB, ANTH, and SR occurred each year in the absence of artiﬁcial inoculum.

TABLE 3: Mean temperature and rainfall for Southwest Michigan Research and
‘ Extension Center for 1990, 1991 and 1992.

 

 

 

 

 

 

Temperature (C)y Rainfall (mm)
Year May Jun Jul Aug Sep May Jun Jul Aug Sep
1990 13.9 20.8 22.0 21.3 18.7 150 83 77 95 155
1991 19.4 23.2 23.8 22.6 17.3 41 91 80 61 81
1992 15.4 18.6 20.7 19.6 16.8 8 45 151 61 75
30 yr 15.1 20.4 22.5 21.7 18.0 94 71 85 95 104
normz

 

 

 

 

 

 

 

 

 

 

 

 

 

Y Temperature data from O’Clare, 12 km north of Niles.
‘ 1951 - 1980
Disease progress of early blight on tomato foliage: Onset of disease varied between

years. For example, at the ﬁrst rating, mean incidence of defoliation due to early blight

(EB) on processing tomato (PRT) plants was 1.4% (n=112), 1.8% (n=56) and 1.9%

 

(n=56) on Aug 16 (day 227) 1990, Jul 17 (day 197) 1991, and Aug 6 (day 218),
respectively (Figure 2). The highest ﬁnal disease rating of PRT plots not sprayed was

25%, 55%, and 87% on Sep 5 (day 247) 1990, Aug 14 (day 225) 1991, and Sep 17

 

 

 

 

72

(day 260) 1992, respectively (Figure 2). The highest ﬁnal disease rating for fresh
market tomato (FMT) plants was 53%, 57%, and 99% on Sep 5 1990, Aug 14 1991,
and Sep 17 1992, respectively (Figure 3). Final ratings of defoliation on Aug 22 (day
233) 1991 are not shown due to an epidemic of bacterial spot (Xanthomonas campestris
pv. vesicatoria) in a number of plots that hampered the ability to rate for BB.

Variance of AUDPC values were not homogenous each year for both PRT and
FMT. Therefore, AUDPC was Log10 transformed prior to analysis of variance and

separation of means. All AUDPC values reported are back-transformed data.

Effect of reduced-s prays and tillage on AUDPC and defoliation due to EB in 1990.
Tillage system and fungicide treatment signiﬁcantly affected disease severity in PRT
plots (Table 4, Figure 2). Zone tillage reduced mean AUDPC values 21% in PRT rows
as compared to CT (Table 5). Application of chlorothlonil reduced defoliation due to
early blight as compared to plots not sprayed (Table 4 and 5). Fifteen weekly
applications of full rate chlorothalonil commencing J une 15 did not provide superior
control as compared to 4 full rate chlorothalonil sprays applied beginning July 11 and
after the accumulation of every 20 DSVs (Figure 2). The main effect (averaged over
CT and ZT plots, n=16) of reduced fungicide rates and applications made after the
accumulation of every 25 DSVs compromised control of defoliation as compared to
plots sprayed weekly (Table 5). However, ﬁnal mean percent defoliation with the most
liberal spray (DSV 25H) was only 12.8% (data not shown).

The interaction between tillage system and fungicide treatment was not
signiﬁcant (Table 4). However, reduced fungicide intergrated with ZT tillage was

additive and of particular interest. For example, using an error mean square of 0.013

 

 

 

 

73
with 80 (if and 8 observations per mean (Table 4), mean separation by LSD test of
AUDPC values demonstrated all TOMCAST based application schedules combined
with ZT provided control comparable to weekly sprays in CT plots (Table 5).

Four plots were affected by a broken ﬁeld tile and estimated values using the
MISVALE ST subroutine of MSTAT-C were substituted. Estimates for pre-transformed
AUDPC values were 95, 103, 186 and 177. Original values were 575, 256, 811 and
966. Weekly estimated values were also substituted to generate disease progress curves.
TABLE 4: Mean squares from analysis of variance for log area under disease progress

curve (AUDPC) for foliar incidence of early blight in processing tomato (PRT) or fresh
market tomato (FMT) in 1990.

 

 

 

 

 

 

 

 

Source AUDPC
of

Variability df PRT FMT
Rep. 7’ 0.052 NS 0.093 NS
Tillage (T) 1 0.327" 0310'
Error a 7 0.022 0.045
Fungicide (F) 6 0.191” 0.475’"
T x F 6 0.010 NS 0.031 NS
Error b 80 and 78z 0.013 0.0172

 

 

 

 

 

 

* ** iii

, , F—test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively. NS, non-
signiﬁcant. '

y 1990 was the ﬁrst year of the experiment resulting in 8 replications per treatment (i.e.
different treatments were not applied to the main plots).

2 4 and 6 values were estimated for plots with poor drainage for processing and fresh

market tomato, respectively, and 1 df for each estimated value was subtracted from the
84 df of the overall residual error mean square.

 

 

74

FIGURE 2: Disease progress curves of percent defoliation of processing tomato plants
estimated weekly in 1990, 1991, and 1992. The insert graph of the 1990 and 1991 ﬁgures
represent disease data of zone tillage (ZT) and conventional tillage (CT) plots transformed
with the logistic model. Values for signiﬁcance of intercept and slope are given in the text.
R- = no rotation; R+ = with rotation

ESTIMATED PERCENT DEFOLIATION ESTIMATED PERCENT DEFOLIATION

ESTIMATED PERCENT DEFOLIATION

75

 

 

 

 

 

 

30_ O/‘Q WEEKLY + or
0". WEEKLY + ZT 1990
0’13 03v 20H + or
l/i st 20H + 21
20- M NO SPRAY + or
M NO SPRAY + 2r
0
I.
10— _-2 :5
CT 5
..-3 O
.l
ZT “4
I 1 5
230 240 250
0 ﬂ

 

 

60-

50-

q

40-
30-

20‘

10-

 

0
190
100-
80-
60-

4o—

20-

 

 

.- -' ' ,
190 200 210 220

I I I I I I I
210 215 220 225 230 235 240 245 250

 

(Dd-'0 WEEKLY + CT
H WEEKLY+ZT 1991
0’13 st 15H + CT
P1 DSV15H + ZT
raw-"i=7 NO SPRAY + or
V? NO SPRAY + ZT

 

 

 

--2
*3
CT ..._4

 

 

I I I I ‘
190 200 210 220 230

T r r l I l "1
195 200 205 210 215 220 225 230

1 992

 
    
  
 
  

WEEKLY DSV 25H NO SPRAY

Mia-set - W
0412-521 '4- V‘V'
O—romaor D'Tl- WW"
0411243 ”'9

    

I I ' I ' I a
230 240 250 260
JULIAN DAY

yyyyyy

 

 

 

76

FIGURE 3: Disease progress curves of percent defoliation of fresh market tomato plants
estimated weekly in 1990, 1991, and 1992. The insert graph of the 1990 and 1991 ﬁgures
represent disease data of zone tillage (ZT) and conventional tillage (CT) plots transformed
with the logistic model. Values for signiﬁcance of intercept and slope are given in the text.
R- = no rotation; R+ = with rotation

 

 

 

 

 

ESTIMATED PERCENT DEFOLIATION

ESTIMATED PERCENT DEFOLIATION

ESTIMATED PERCENT DEFOLIATION

 

 

 

 

 

 

 

 

 

 

   
  
  
  
  
  
 
  
 
   

 

 

 

 

 

 

 

 

 

 

77
60-: O/O WEEKLY .. or 1990
J M WEEKLY + zr
50‘ M osv 20H + cr
1 V'- st 20H + ZT
40‘ M no SPRAY + CT
‘ t—r’y NO SPRAY + 21
30a _0
1 r4 X
. ‘3 8
zr I .1
10a ”4
s
I go :10 250 -
C I T I ‘lﬁ T T— I I
210 215 220 225 230 235 240 245 250
60W 0",0 WEEKLY*CT 1
M WEEKLY ‘27
I 0’11 st 15H ocr 1 991
50‘ H osvrsmzr
‘ M NO smuwcr
40~ v1 no SPRAY+zr
1 I"°
30- M
X
/ ”'2 '3
o
20- c, N 9.
I-J
1 T— T T— ‘5
10" 190 200 210 220 m
c *‘T”
7 ‘ 7— I T I T I I
1 90 195 200 205 210 2‘1 5 220 225 230
1001
1 992
80-
WEEKLY osvzsu
0’0 meet 0"‘11 .
60“ v. 3.5:? M .
. o. '0 R¢&CT 0- ‘D .
.— ‘. 3.521 I-- ‘I .
40'-
’0
20-
0 “2"",— r f

‘ T I
1 90 200 21 0 220 230

r ' 1 ' 1
240 250 260
JULIAN DAY

*1

 

 

78

TABLE 5: Backtransformed 1990 values for area under the disease progress curve for
processing tomato and fresh market tomato. Analysis of variance and mean separation
was based on log transformed data.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PROCESSING TOMATO
FUN GICIDE TILLAGE FUNGICIDE
TREATMENT TREATMENT TREATMENT

CTp ZT MEANq
WEEKLY 74 xyz 64 z 69 d
DSV 20L 97 86 xy 91 bc
DSV 20H 95 x 71 yz 83 cd
DSV 25L 110 89 xy 100 bc
DSV 25H 118 96 x 107 b
DSV 20H 110 70 yz 90 c
NO SPRAY 130 132 156 a
MEAN OF TILLAGE 108 85

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FRESH MARKET TOMATO

CT" ZT MEANq
WEEKLY 94 z 96 z 95 d
DSV 20L 140 121 z 130 c
DSV 20H 186 121 z 152 be
DSV 25L 200 136 168 b
DSV 25H 149 134 142 be
DSV 20H 141 125 z 133 c
NO SPRAY 443 263 355 a
MEAN OF TILLAGE 172 135

 

 

 

 

P values are means of 8 replications. xyz is seperation of selected means within each
tomato type by the Least Signiﬁcant Difference (LSD) test, P = 0.05.

‘1 values are means (n=16) of conventional tillage (CT) and zone tillage (ZT) 101$-
a-d rs mean seperation within columns of each tomato type by LS , = 0%5.

 

 

 

 

 

79

Tillage system and fungicide treatment signiﬁcantly affected disease severity in

FMT plots (Table 4, Figure 3). Zone tillage reduced mean AUDPC values 22% in FMT
rows as compared to CT (Table 5). Fungicide treatment reduced AUDPC values
compared to plots not sprayed (Table 5). The main effect (n=16 per mean) of weekly
applications was superior to each main effect of TOMCAST based treatments.
However, mean separation by the LSD test of individual treatments (n=8 per mean)
demonstrated reduced and full rates of chlorothalonil applied after the accumulation of
every 20 DSVs and integrated with ZT offered control equal to weekly applications in
CT and ZT plots (Table 5). Figure 3 highlights the disease progress curve of the DSV
20H x ZT treatment which is not signiﬁcantly above disease progress curves generated
from plots sprayed weekly.

Six estimated FMT AUDPC values - 123, 114, 215, 163, 431, and 480 replaced
pretransformed outliers of 978, 950, 950, 768, 1352, and 1160, respectively.

In summary for 1990, ZT decreased severity of defoliation due to early blight
when tomato was planted in plots with no recent history of tomato. In ZT plots,
chlorothalonil applied to PRT and FMT after the accumulation of every 25 DSVs and
20 DSVs, respectively, provided control comparable to conventional production systems
(weekly fungicide applications and conventional tillage). This represented 80% and
73 % fewer fungicide applications without signiﬁcantly compromising percent

defoliation.

Effect of reduced-s prays and tillage on AUDPC and defoliation due to EB in 1991.
Tomato transplants were planted in ZT plots on the same row center as in 1990.

Overwintered tomato fruit skins and dead vines were prevalent on the soil surface.

 

 

 

80

Minimal surface debris was apparent in CT plots. Commencing June 13, 11 weekly
sprays of chlorothalonil were applied (Table 2). Six (DSV 15) and 4 (DSV 20)
applications for the reduced-sprays program were initiated commencing June 26 after
the accumulation of 35 DSVs, according to the TOMCAST model. Fruit were ”walnut
size” on FMT plants and just begirming to set on PRT plants on June 26.

Tillage system and fungicide treatment signiﬁcantly affected disease severity in
PRT plots (Table 6, Figure 2). Mean AUDPC values in PRT CT plots were 69% of
mean AUDPC values in ZT plots (Table 7). Plots sprayed with chlorothalonil had
signiﬁcantly lower AUDPC values as compared to plots not sprayed (Table 7 ). Weekly
fungicide applications provided superior control of defoliation as compared to all other
treatment combinations (Table 7). No treatment combination provided control
comparable to the conventional system, with weekly applications of fungicide and
conventional tillage. Final percent defoliation recorded on Aug 14 was 14.4%, 15.3%
and 16.0% for the weekly, DSV 15H and DSV 20H treatments, respectively.

Tillage system and fungicide treatment also signiﬁcantly affected disease
severity in FMT plots (Table 6, Figure 3). Mean AUDPC values in FMT CT plots were
69% of mean AUDPC values in ZT plots (Table 7), similar to PRT results. Plots
sprayed with chlorothalonil had signiﬁcantly lower AUDPC values as compared to
plots not sprayed (Table7). Mean AUDPC values for main effects (n=8 per mean) of
plots sprayed weeldy were not signiﬁcantly different than values calculated from plots
sprayed with full rate chlorothalonil after the accumulation of every 15 DSVs (Table
7). Other TOMCA ST-based spray treatments compromised control as compared to the
weeldy treatment (Table 7). However, mean separation by the LSD test of individual

treatments (n=4 per mean) demonstrated reduced and full rates of chlorothalonil applied

 

 

 

 

81

after the accumulation of every 15 and 20 DSVs and combined with CT offered control
equal to weekly applications in CT plots (Table 7).

In summary for 1991, tomato (1991) planted after tomato (1990) allowed for
signiﬁcant levels of defoliation in ZT plots. Conventional tillage apparently reduced
initial levels of inoculum. In FMT plots, CT functioned in an additive manner when
integrated with reduced fungicide applications to control defoliation equal to plots
sprayed weekly. A reduced fungicide spray schedule did not provide equal control of
defoliation in PRT plots as CT plots sprayed weekly.

TABLE 6: Mean squares from analysis of variance for log area under disease progress

curve (AUDPC) for foliar incidence of early blight in processing tomato (PRT) or fresh
market tomato (FMT) in 1991.

 

 

 

 

 

 

 

 

Source of AUDPC
Variability df
PRT FMT

Rep. 3y 0.062 NS 0.191 NS
Tillage (T) 1 0.372" 0.354"
Error a 3 0.011 0.010
Fungicide (F) 6 0280*" 0.289”
T x F 6 0.017 NS 0.026 NS
Error b 36 0.013 0.014

 

 

 

 

 

 

1‘ it ***

, , F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively. NS, non-
signiﬁcant.

’ in 1991 tomato was planted to half the plots (continuous tomato main plots, n=56) and
cucumber to the other half (main plots with rotation).

’ plots with poor drainage were not located in the continuous tomato main plot treatments.

 

 

 

82

TABLE 7: Backtransformed 1991 values for area under the disease progress curve for
processing tomato and fresh market tomato. Analysis of variance and mean separation
was based on log transformed data.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PROCESSING TOMATO
FUNGICIDE TILLAGE TREATMENT FUN GICIDE
TREATMENT CI‘p ZT TREATMENT
MEANq
WEEKLY 103 z _ 204 wxy 154 c
DSV 15L 199 wxy 306 252 b
DSV 15H 172 xy 260 w 216 b
st 20L 180 NY 272 W 226 b
st 20H 165 Y 245 W" 205 b
NO SPRAY 479 469 474 a
NO SPRAY 378 516 447 a
MEAN OF TILLAGE 212 308 _=
FRESH MARKET TOMATO
mama mMSETEREW wearer.
MEANq
WEEKLY 268 z 395 xy 332 c
DSV 15L 313 yz 588 w 450 b
DSV 15H 325 yz 433 z 379 be
DSV 20L 375 yz 579 wx 477 b
DSV 20H 306 yz 632 w 469 1)
NO SPRAY 929 v 927 v 928 a
NO SPRAY 940 v 1042 v 991 a
MEAN OF TILLAGE 430 621
P values are means of 4 replications. v-z is seperation of selected means within each
tomato type by the Least Signiﬁcant Difference (LSD) test, P = 0.05.
‘1 values are means (n= 8) of conventional tillage (CT) and zone tillage (ZT lots.
a—c is mean seperation within each column of tomato type by LSD, P = . 5.

 

 

 

 

83

Effect of reduced-sprays, tillage and rotation on AUDPC and defoliation due to
EB in 1992. All plots were conventionally tilled in 1991 and the 2-year ZT cycle
(Figure 1) was re-initiated in the early spring. Late season forecasted frosts delayed
time of planting to Jun 3 and Jun 10 for FMT and PRT, respectively. Cool weather
persisted through the spring and DSVs cumulated slowly, with average T° frequently
below the 13°C threshold (Table 1) during periods when leaves were wet (e.g. night
temperatures dipped to a low of 2°C June 22). The PRT experiment was terminated
pre-maturely on Sep 29, 1992 to avoid forecasted frost and loss of ﬁnal yield data.

Commencing June 25, 13 weekly sprays of chlorothalonil were applied (Table
2). Initial application of TOMCAST-based sprays was scheduled for the ”safe date” of
Jul 11 but wet weather delayed the ﬁrst spray until Jul 16. Five, 4 and 3 subsequent
applications were scheduled after the accum-ulation of every 15 (DSV 15L), 20 (DSV
20L) and 25 (DSV 25H) disease severity values. Plots scheduled to receive the full rate
of chlorothalonil after the accumulation of every 15 (DSV 15H) or 20 (DSV 20H)
DSVs were inadvertently not sprayed Jul 16. In the latter treatments, a total of 4 and 3
sprays were applied commencing Jul 30.

With only 1 df for the denominator and numerator, signiﬁcant effects due to
rotation were not observed (Table 8) even though mean AUDPC in rotation PRT plots
was 70% of values in plots not rotated (Table 9). The main effect of CT as compared
to ZT was also not signiﬁcant but the rotation x tillage interaction was (Table 8). Zone
tillage decreased mean AUDPC values from 696 to 479 percent-days in plots planted to
continuous tomato. In contrast, mean AUDPC values increased from 368 percent-days
in CT plots to 446 in ZT plots when combined with rotation (Table 9). No other

interactions were signiﬁcant (Table 8).

 

 

84

TABLE 8: Mean squares from analysis of variance for log area under disease progress
curve (AUDPC) for foliar incidence of early blight in processing tomato (PRT) or fresh

market tomato (FMT) in 1992.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Source AUDPC
of

Variability PRT FMT
Rep. 1y 0.060 NS 0.137 NS
Rotation (R) 1 0.330 NS 0.097 NS
Error a 1 0.007 0.013
Tillage (T) 1 0.022 NS 0.006 NS
R x T 1 0.211' 0513'
Error b 2 0.004 0.007
Fungicide (F) 6 0.204m 0.274'"
R x F 6 0.002 NS 0.007 NS
T x F 6 0.005 NS 0.003 NS
R x T x F 6 0.017 NS 0.019 NS
Error c 24 0.009 0.010

 

 

 

 

 

2",” F-test signiﬁcant at P - 0.05, P - 0.01 or P = 0.001, respectively. NS, non-
signiﬁcant.

3' nearly all sub-sub-plot treatments in 2 replications of a sub-plot treatment (conventional
tillage) of a main-plot treatment (tomato rotated to cucumber) were adversly affected by
a ﬁeld tile that malfunctioned, resulting in poor drainage. Therefore the data were
analyzed over 2 replications instead of 4 (n=56, not 112).

 

 

85

 

TABLE 9: Backtransformed 1992 values for area under the disease progress curve for
processing tomato and fresh market tomato. Analysis of variance and mean separation
was based on log transformed data.

 

 

 

 

 

 

 

 

 

 

 

PROCESSING TOMATO

FUNGICIDE NO ROTATION ROTATION FUNGICIDEq

TREATMENT CT ZT CT ZTp TRMT MEAN
WEEKLY 366 w-z 332 xyx 234 z 250 yz 295 c
DSV 15L 859 422 wx 320 xyz 585 546 b
DSV 15H 560 481 wx 377 wxy 402 wx 465 b
DSV 20L 794 437 wx 416 wx 448 wx 524 b
DSV 20H 614 419 wx 371 wxy 378wxy 445 b
DSV 25H 565 w 446 wx 357 xyz 351 xyz 430 b
NO SPRAY 528 1054 590 1005 1044 a
MEAN OF COLUMN 696 479 368 446
MEAN OF ROTATION 577 405
MEAN OF TILLAGE CT = 506 ZT = 462 ‘

 

 

 

 

FRESH MARKET TOMATO

 

 

 

FUNGICIDE NO ROTATION ROTATION FUNGICIDEq

TREATMENT CT ZT CT ZTp TRMT MEAN
WEEKLY 516 xy 406 xyz 309 z 330 gz 390 c
DSV 15L 1483 550 x 450 xyz 100 873 b
DSV 15H 877 679 504 xy 830 722 b
DSV 20L 1047 608 x 652 x 849 789 b
DSV 20H 857 541 x 533 x 721 663 b
DSV 25H 826 490 xyz 512 xy 741 642 b
NO SPRAY 2291 1552 1099 1722 1666 a

 

MEAN OF COLUMN 1019 ‘ . 626 542 804

 

 

MEAN OF ROTATION 799 660

 

 

 

 

 

MEAN OF TILLAGE CT = 743 ZT = 710

 

 

P values are means of 2 replications. w-z is seperation of selected means within each
tomato type by the Least Signiﬁcant Difference (LSD) test, P = 0.05 .

q values are means (n= 8) of conventional tillage (CT) and zone tillage (ZT) plots averaged
over rotation treatment. a-c is mean seperation within each column of tomato type by

LSD, P = 0.05.

 

 

 

 

 

86

Fungicide treatment signiﬁcantly affected disease severity (Table 8). Plots
sprayed with fungicide decreased disease severity compared to plots not sprayed and
mean AUDPC values of plots sprayed weekly were signiﬁcantly lower than all
TOMCA ST-based treatments. However, Table 9 and Figure 2 highlight the integrated
effect of reduced fungicide applications when combined with rotation. The DSV 25H
treatment combined with rotation provided control comparable to plots sprayed weeldy
in CT and ZT plots.

In the case of fresh market tomato plants, numerous plots not sprayed
approached 100% defoliation. Rotation decreased mean AUDPC values 17% but this
was not signiﬁcant (Tables 8 & 9). Tillage also did not affect disease severity but the
interaction of rotation x tillage was important (Table 8) similar to results observed for
PRT. Fungicide application reduced disease severity as compared to plots not sprayed.
TOMCA ST-based sprays signiﬁcantly controlled defoliation as compared to plots
sprayed weekly (Table 9, Figure 2) but did not provide equal control as CT plots

sprayed weekly and rotated to cucumbers.

Effect of fungicide and tillage on rate of disease progress. The logistic and
Gompertz model both accounted for a high percentage of variation in the incidence of
defoliation of tomato plants. For example, coefﬁcients of determination (R2) for each
plot of PRT with ﬁve temporal observations in 1991 ranged from 0.88 to 0.99. Mean
Q SD) R2 was 0.966 1 0.027 and 0.961 1 0.034 for the logistic and Gompertz model,
respectively, and plots of residuals conﬁrmed the acceptability of both models. The
logistic model was used for all analyses in 1990 and 1991. Variance increased with

means in 1992 and simple models did not provide good ﬁt and data were not

 

 

 

 

87
transformed.

In 1990, the rate (r) of disease progress in PRT plots not sprayed was 0.14
logits day‘1 as compared to 0.10 (weekly), 0.12 (DSV 20L), 0.11 (DSV 20H), 0.13
(DSV 25L), and 0.12 (DSV 25H). The rate (r) of disease progress in FMT plots not
sprayed was 0.15 logits day'1 as compared to 0.10 (weekly), 0.10 (DSV 20L), 0.08
(DSV 20H), 0.12 (DSV 25L), and 0.11 (DSV 25H). Mean epidemic rates in CT plots
as compared to ZT plots were 0.12 vs 0.11 (P=0.3) and 0.12 vs 0.10 (P=0.08) for PRT
and FMT, respectively (Figures 2 & 3, insert). The level of the regression line was
higher in CT compared to ZT plots in both cases (P = 0.001).

In 1991, the rate of disease progress for untreated PRT was 0.13 in plots not
sprayed and 0.08, 0.11, 0.08, 0.09 and 0.09 for the weeldy, DSV 15L, DSV 15H, DSV
20L and DSV 20H treatments, respectively. The rate of disease progress for untreated
FMT was 0.13 as compared to 0.08 and 0.09 to 0.10 in weekly or TOMCAST-based
treatments. Mean ZT curves were higher (P=0.001) for both PRT and FMT. Epidemic
rates in PRT and FMT CT plots compared to ZT plots were 0.11 vs 0.10 (P=0.13) and

0.11 vs 0.09 (P=0.05), respectively (Figures 2 & 3, insert).

Early season incidence of disease. In 1991, plants in ZT plots acquired leaf lesions
within 1 wk of ﬁeld setting (Table 10). No conclusive isolation was obtained. Plants in
ZT plots, and in the absence of rotation, also developed collar rot (Table 10), caused
by Altemaria solani, apparently in response to high early-season day time temperatures
(e. g. up to 33°C). In many cases the lesion girdled the entire stem at the soil line but
early plant productivity did not appear to be affected. For example, mean PRT plant

height on Jun 19 was greater (P=0.05) in ZT plots (28.6 cm) as compared to plants in

 

 

 

 

88

Cl‘ plots (26.5 cm). FMT plants were 39.8 and 36.2 cm (P=0.017) in ZT and CT plots,

respectively.

TABLE 10. Percent plants with early season incidence (1 SE) of a leaf spot and
Altemaria solani collar rot on processing and fresh market tomato plants, 1991.

 

 

 

 

 

 

 

 

 

 

 

Date and Tillage Tomato Type
Disease Treatment
Processing Fresh Market
May 30 CT 5.7 1 1.7 0.0
Leaf Spot
ZT 7.3 1 2.2 7.8 1 2.2
June 6 Cl‘ 1.11 0.5 7.6 1 1.8
Leaf Spot
21‘ 50.2 1 5.7 89 1 3.0
June 19 Cl‘ 0.11 0.1 1.11 0.6
Collar Rot
ZT 8.1 1 1.5 40.7 1 3.7

 

 

 

On Jul 16 1992, incidence (percent plants) and severity (mean no. lesions per
plant 1 SE) of EB lesions was assessed in weekly, DSV 15H and no spray PRT plots
which had been sprayed 3, 0, and 0 times, respectively. Mean percentage of plants with
lesions was 7.5 1 1.9, 42.2 1 9.0 and 44.4 1 8.3% in weekly, DSV 15H and no spray
plots, respectively. In Cl‘ plots, incidence was 23.3 1 4.8% as compared to 39.4 1
8.0% in ZT plots. The mean effect of rotation was to reduce early initial incidence
from 49.0 1 7.3% to 13.8 1 3.3%. Incidence and severity were highly correlated and

could be described by an additive exponential model (Figure 4). Lesions were small

(<5 mm) and limited to 1 to 12 per plant.

 

 

 

 

89

 

..A
h.)

RE

(I)
1 __I _.J
1* 1—1—«gr—rﬁqrm

SEVERITY
AVERAGE NO. LESIONS PER PLANT
T
we

4 3101151!
31!
3101!

 

 

 

C ’4"— r 1 T I ' . ‘ I ‘
0 20 40 60 80 100
INCIDENCE
PERCENT PLANTS WITH EB LESIONS

FIGURE 4:Incidence and severity of early blight symptoms recorded on processing
tomato plants on July 16, 1992.
Effect of reduced-s prays, tillage and rotation on fruit mold incidence. Sub-samples
of PRT fruit and all harvested FMT fruit were evaluated for symptoms of EB, ANTH,
and SR without regard for severity of symptoms. The highest recorded incidence of
PRT fruit mold in plots not sprayed was 20.5%, 24.0% and 52.0% in 1990, 1991, and
1992, respectively. The highest values recorded for FMT fruit mold incidence was
22.2%, 20.2%, and 19.5% in 1990, 1991, and 1992, respectively. Incidence of EB,
ANTH and SR expressed as a proportion of fruit with mold symptoms was calculated
for each year (TABLE 11 A & B).
Soil rot was the primary mold on FMT fruit in 1990 but the proportion steadily

declined each year and EB became the primary mold problem. The proportion of each
mold changed over time but was not dramatically affected by tillage nor rotation. In

1990, proportion of ANTH was 0.07 in FMT fruit harvested from ZT plots compared

 

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91

to 0.12 in CT plots (P=0.09). Processing tomato 1992 had a higher proportion of SR in
ZT plots (proportion = 0.12) as compared to CT plots (proportion = 0.07) in the
absence of rotation. In contrast, proportion SR was lower in ZT plots (proportion =
0.10) as compared to CT plots (prOportion = 0.25) after rotation. In 1992, proportion
ANTH on PRT and FMT fruit decreased when sprayed with fungicide compared to
plots not sprayed (data not shown). In contrast, proportion SR increased when fruit

were sprayed weekly as compared to a reduced or no-sprays program.

Percent fruit mold incidence in 1990. Tillage did not signiﬁcantly affect incidence of
PRT fruit mold (Table 12). Fungicide treatment did signiﬁcanlty affect PRT fruit mold
incidence (Table 12, Figure 5). Full rate fungicide applications after the accumulation
of every 20 (4 sprays) and 25 (3 sprays) DSVs limited mold incidence comparable to
the weekly (15 sprays) treatment. The main effects (means over Cl‘ and ZT plots) are
shown in Figure 5 but the integrated effect of fungicide and tillage was of particular
interest. For example, ZT plots sprayed with full rate chlorothalonil after the
accumulation of every 20 or 25 DSVs limited fruit mold incidence to 4.1% and 5.6%,
respectively. This was not signiﬁcantly different as compared to 3.5% and 3.4% in CT
and ZT plots sprayed weekly. The DSV 20 and DSV 25 treatments using reduced
fungicide rates compromised disease control.

Tillage did not affect incidence of fruit mold in FMT plots (Table 12, Figure 6).
Fungicide treament had a signiﬁcant affect on fruit mold incidence but reduced-sprays
did not provide control of fruit mold in FMT plots as compared to plots not sprayed
(Table 12, Figure 6). Soil rot, the predominant mold, did not appear to be effectively

controlled except by weekly applications of chlorothalonil.

 

 

 

 

92

TABLE 12: Mean squares from analysis of variance for percent fruit with mold due to
early blight, anthracnose or soil rot in processing tomato (PRT) or fresh market tomato

 

 

 

 

 

 

 

 

(FMT) in 1990.
Source % Fruit with Mold

Varizlfaility df PRT FMT
Rep. 3y 258.1 NS 126.0 NS
Tillage (T) 1 280.7 NS 1.4
Error a 3 149.4 24.8
Fungicide (F) 6 140.8” 1340*"
T x F 6 13.0 NS 19.3 NS
Error b 36 17.0 21.7

 

 

 

 

 

 

* ii iii

, , F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
NS, non-signiﬁcant.

’ fruit were harvested only from main plots destined to be in continuous tomato (n=56).
Harvested PRT fruit was returned to each plot after evaluation and harvested FMT fruit
was removed from all plots.

1 plots with poor drainage were not located in the continuous main plot treatments.

Percent fruit mold incidence in 1991. Fungicide signiﬁcantly affected PRT fruit mold
incidence in 1991 and tillage did not (Table 13, Figure 5). Fungicide applied at the
high rate and low rate after the accumulation of every 15 (6 sprays) or 20 (4 sprays)
DSVs provided control comparable to the weekly (11 sprays) treatment. This
represented 45% to 64% fewer fungicide applications. The tillage x fungicide
interaction was not signiﬁcant (Table 13). Tillage signiﬁcantly affected FMT fruit mold
incidence (Table 13). CT reduced FMT mold incidence as compared to ZT (Figure 6).

Main effects of the DSV 15H and DSV 20H treatments were not signiﬁcantly different

 

 

 

93

than the main effect of weekly applications (Figure 6). Tillage x fungicide interaction
was not signiﬁcant (Table 13). However, the integrated effect of reduced sprays and
CI‘ in 1991 was additive and provided control of fruit mold most comparable to plots
sprayed weekly. For example the CT x DSV 15H and CT x DSV 20H treatments had a
total mold incidence of 7.8% and 7.6% as compared to 5.5% and 6.8% in CI‘ and ZT
plots sprayed weekly.

TABLE 13: Mean squares from analysis of variance for percent fruit with mold due to
early blight, anthracnose or soil rot in processing tomato (PRT) or fresh market tomato

 

 

 

 

 

 

 

 

(FMT) in 1991.
Source of % Fruit with Mold
Variability
df PRT FMT
Rep. 3’ 95.8 NS 58.3 NS
Tillage (T) 1 236.2 NS 507.0 "
Error a 3 251.4 12.3
Fungicide (F) 6 112.4' 108.6“
T x F 6 45.0 NS 15.5 NS
Error b 361 40.8 20.2

 

 

 

 

 

 

* it *i’*

, , F—test signiﬁcant at P = 0.05, P - 0.01 or P = 0.001, respectively.
NS, non- signiﬁcant.

’ in 1991 tomato was planted to half the plots (continuous tomato main plots, n=56)
and cucumber to the other half(tomato rotated to cucumber main plots).

2 plots with poor drainage were not located in the continuous tomato main plot
treatments.

 

94

FIGURE 5: Incidence of fruit mold expressed as percent of total weight of processing
tomato fruit harvested in 1990 and 1991. Bars represent the main effect of each
treatment (i.e. mean of CT and ZT plots combined). Bars with the same letter are not
signiﬁcantly different based on protected LSD value. P value indicates the level of
signiﬁcance between the main effect of CT as compared to ZT on incidence of fruit

mold.

 

% FRUIT WITH MOLD

% FRUIT WITH MOLD

 

 

 

 

 

 

 

  
    
 

  
 

 
    
    
   
 
  
  
 

25 ‘
%EB 1990 =Ns
20 %ANTH
A
%SR
15
s E
10 B BC E
BC E =
,1 IIIIII IIIIII IIII IIIIII II I IIIIII
. WEEKLY . osvon . DSV25H . or ZT
st 20L DSV 25L NO SPRAY
FUNGICIDE TREATMENT TILLAGE
25
%EB
20 : %ANTH
%SR
15
10
5 E g
0 I I

    

 

 

. DSV15H . DSVZOH
15L

st 20L NO SPRAY

FUNGICIDE TREATMENT

  
  

 

96

FIGURE 6: Incidence of fruit mold expressed as percent of total weight of fresh market
fruit harvested in 1990 and 1991. Bars represent the main effect of each treatment (i.e.
mean of CT and ZT plots combined). Bars with the same letter are not signiﬁcantly
different based on protected LSD value. P value indicates the level of signiﬁcance
between the main effect of CT as compared to ZT on incidence of fruit mold.

 

% FRUIT WITH MOLD

% FRUIT WITH MOLD

     
 

“5 1990 I P=NS

  
 

% ANTH
% SR

    

01

    

or ZT
TILLAGE

WEEKLY DSV 20H . DSV 25H
DSV 20L DSV 25L NO SPRAY

FUNGICIDE TREATMENT

 

 

h.)
(.11

 
 

% EB
% ANTH
% SR

 
    
   

15

   
  

20H .
DSV 20L NO SPRAY
FUNGICIDE TREATMENT TILLAGE

WEEKLY . DSV15H . DSV
DSV15L

 

 

 

98

Twenty fully ripe No. 1 symptomless fruit were collected from the weekly,
DSV 20H, and no spray treatment on three harvest dates to determine the incidence of
latent anthracnose infections and the potential market-life of harvested fruit. After one
week at ambient air temperatures, over 30% of the fruit not sprayed had anthracnose
lesions (Table 14). Overall, the weekly and DSV 20H treatment limited latent
infections as compared to the no spray treatment. Conventional tillage signiﬁcantly
limited incidence of anthracnose on fruit as compared to ZT (Table 14). Effect of
harvest date and interactions were not signiﬁcant (data not shown).

TABLE 14. Post-harvest incidence (%) of fully red N 0.] fresh market tomato fruit
with symptoms of anthracnose in 1991.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TREATMENT HARVEST DATE
AUG 5 AUG 12 AUG 21 MEAN
WEEKLY 11.3 by 15.4 b 24.5 a 17.1 b
DSV 20H 13.3 b 16.5 b 14.0 b 14.6 b
NO SPRAY 32.4 a 38.3 a 34.6 a 35.1 a

MEAN 19.0 23.4 24.4

EFFECT OF TILLAGE
CONVENTIONAL TILLAGE 19.0
ZONE TILLAGE 25.5 (P = 0.05)

 

 

 

 

" Values are means of three harvest dates with 20 fruit collected at harvest from 4 Cl‘
plots and 4 ZT plots (n=8 plots, 160 fruit).

’ within column values followed by the same letter are not signiﬁcantly different at
P=0.05. Harvest date and any interaction was not signiﬁcant.

2 main effect of tillage averaged over all harvest dates and fungicide treatments.

 

 

 

99

Percent fruit mold incidence in 1992. The rotation x tillage and rotation x tillage x
frmgicide interactions were highly signiﬁcant for both PRT and FMT in 1992 (Table
15). In general, mold incidence decreased in ZT x no—rotation plots and increased in ZT
x rotation plots as compared to CT. The effect in no spray plots was most pronounced
(Figure 7 ).

The PRT experiment was prematurely terminated and most fruit were not fully
ripe. On average, fungicide applied at the full rate after the accumulation of every 25
DSVs (3 sprays) provided PRT mold control comparable to weekly (13 sprays)
applications and this was inﬂuenced by cultural practice (Figure 7 ). For PRT plots, the
DSV 25H treatment was most Similar to the weekly treatment when combined with ZT -
or rotation (Figure 7). The DSV 15 and DSV 20 treatments with full rate chlorothalonil
did not provide similar control of fruit mold as compared to plots sprayed weekly (data
not shown). The initial spray for theSe treatments was delayed 14 days Jul 30 (Table 2)
as compared to other TOMCAST-based treatments. This observation, combined with
the effectiveness of the DSV 25 full rate treatment suggests that a reduced fungicide
schedule is more effective than eliminating funeicide sprays early in the season.

The main effect of all TOMCAST-based treatments except the DSV 15L
provided FMT fruit mold control comparable to the weekly treatment (data not shown).
The DSV 25H treatment, the most liberal fungicide schedule, was equally effective in
controlling FMT mold incidence for all treatment combinations as compared to plots
sprayed weekly (Figure 7). Plots not sprayed had the highest overall incidence of mold
(P=0.05). Figure 7 highlights the weekly, DSV 25H and no spray treatment and their

interaction with rotation and tillage.

 

 

 

 

100

TABLE 15: Mean squares from analysis of variance for percent fruit with mold due to
early blight, anthracnose or soil rot in processing tomato (PRT) or fresh market tomato

 

 

 

 

 

 

 

 

 

 

 

 

 

(FMT) in 1992.
Source AUDPC
of

Variability df PRT FMT
Rep. 1’ 1193.3 NS 9.8 NS
Rotation (R) 1 494.6 NS 23.8 NS
Error a 1 514.2 42.4
Tillage (T) 1 6.4 NS >0.1 NS
R x T 1 1767.6" 113.54"
Error b 2 11.1 0.8
Fungicide (F) 6 789.8” 62.8""
R x F 6 66.9 NS 4.2 NS
T x F 6 40.6 NS 2.5 NS
R x T x F 6 2250* 158*
Error c 24 69.4 5.5

 

 

 

 

 

 

* ** ***

, , F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
NS, non-Signiﬁcant.

’ nearly all sub-sub-plot treatments in 2 replications of a sub-plot treatment (conventional
tillage) of a main-plot treatment (tomato rotated to cucumber) were adversly affected by
a ﬁeld tile that malfunctioned, resulting in poor drainage. Therefore the data were
analyzed over 2 replications instead of 4 (n=56, not 112).

 

 

FIGU
(tor)
betwe
of 2 1
a one

 

 

 

101

FIGURE 7: Incidence of fruit mold expressed as percent of total weight of processing
(top) and fresh market (bottom) tomato fruit harvested in 1992. The 3 way interaction
between rotaton, tillage and fungicide treatment was signiﬁcant. Each point is the mean
of 2 replications in conventional tillage (CT) or zone tillage (ZT) plots combined with
a one year rotation or not rotated.

 

0J0.)— ILL>> 1.1—3W.“— IrszW—wn— 0. .CE ILL\<r LI: .0” LI.<1|L!\H.LE

 

PERCENT FRUIT WITH MOLD

PERCENT FRUIT WITH MOLD

 

102

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

60— PROCESSING TOMATO

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40 ‘ —e— DSV 25 H

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60—
FRESH MARKET TOMATO
504
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—-v— NO SPRAY

30~

1
204 v

V

.7 \ /
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Ellec
yield
PRT
not 1

com]

inciI

iJlP

All

we]

obT.

AL

001

 

 

103

Effect of defoliation due to EB on total yield and incidence of fruit mold. Total
yield in plots not sprayed were not signiﬁcantly reduced due to defoliation for both
PRT and FMT in 1990 and 1991 (Chapter 3). In 1992, lack of fungicide application did
not impact FMT yield but PRT yield in plots not sprayed was reduced 25% as
compared to plots sprayed with Bravo (Chapter 3, marginally signiﬁcant at P = 0.06).

The incidence of fruit mold caused by EB was affected by AUDPC (non
transformed values) (Figure 8). Based on analyses of data collected from 1990 to 1992,
incidence of EB fruit mold increased 1.4% to 2.3% for each 100 percent-days increase
in PRT AUDPC. The relationship between FMT incidence of fruit mold due to BB and
AUDPC varied more among years and was not as convincing. For 1990 to 1992, the
linear equation estimates varied between 0.3% to 1.2% increase in EB fruit mold with
each 100 percent-days increase in AUDPC. In 1992, ANTH fruit rot increased 0.2% in
FMT plots with each 100 percent—days increase in AUDPC. No relationship was
obvious in 1990 and 1991, nor in PRT plots. Incidence of SR was not related to
AUDPC values (data not shown).

Incidence of fruit mold due to BB and ANTH was correlated (Table 16). The
correlation coefﬁcient and slope remained relatively constant among years for FMT.
The coefﬁcient of correlation for all values over the 3 yr was 0.44 and the incidence of
ANTH was 0.25 the incidence of EB. In PRT, the correlation was signiﬁcant in 1990

and 1991 but not 1992. The overall correlation coefﬁcient was not signiﬁcant.

 

 

FIGURI
anthracr
(AUDP'
provide
determi
and Al

 

 

104

FIGURE 8. Relationship of incidence Of fruit mold, due to early blight (top) or
anthracnose (bottom), to non—transformed Area Under the Disease Progress Curve
(AUDPC) values in processing tomato (left) and fresh market tomato (1i ght). Equations
provide the estimated intercept, slope, error mean sauare (EMS) and coefﬁcient of

determination (R2) for linear regression analysis between early blight or anthracnose
and AUDPC values.

 

 

 

 

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106

TABLE 16: Pearson's correlation coefﬁcient (r), intercept, slope, standard error (SE) of
the slope and signiﬁcance value of the correlation of the relationship between
incidence of fruit mold due to early blight and anthracnose in processing (PRT) and
fresh market tomato (FMT) in 1990, 1991, and 1992.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TREATMENT r INTERCEPT SLOPE SE OF P
SLOPE VALUE
1990 PRT 0.463 0.12 1.155 0.301 0.001
1991 PRT 0.402 1.61 0.543 0.177 0.003
1992 PRT NS
1990 TO 1992 0.088 3.38 0.075 0.067 0.262
PRT
1990 FMT 0.396 -0.03 0.316 0.10 0.002
1991 FMT 0.494 0.14 0.293 0.071 0.001
1992 FMT 0.567 -0.64 0.258 0.051 0.001
1990 TO 1992 0.437 -0.11 0.275 0.044 0.001
FMT

 

 

 

 

 

I
with red
tomato
with co
and we.
minimi:
practice
inputs 1
overall
and Ht
Challer

the be]

Probie
System
in th'r
rOtatit
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comp;
IteaH
Alton

Ieacti

 

 

107
DISCUSSION

Integrated control of early blight (EB), anthracnose (ANTH), and soil rot (SR)
with reduced fungicide input was possible in a fresh market (FMT) and a processing
tomato (PRT) production system. However, the dynamics of disease incidence varied
with culture practice, reduced sprays, tomato type and year. Conventional tillage (CT)
and weekly application of full rate fungicide required less management and appeared to
minimize crop quality risk as compared to reduced-sprays and conservation tillage
practices. However, conservation tillage, use of cover crops and reduced fungicide
inputs have considerable beneﬁt for soil health, soil quality, environmental quality and
overall long-term sustainability of ﬁeld level productivity and proﬁtability (Coolman
and Hoyt 1993; Fretz et a1. 1993; Morse 1993; Sarrantonio 1992; Stephens 1990). The
challenge is to combine the beneﬁts of reduced fungicide usage without compromising
the beneﬁts of conservation tillage and cover crop use.

Zone tillage (ZT) is a form of conservation tillage designed to circumvent
problems of reduced yield or plant stand associated with complete no-till production
systems (Doss et al. 1981; Price and Baughan 1988). The zone tillage system employed
in this study enhanced or did not affect tomato total yields when combined with
rotation (Chapter 3, Grajauskis 1990). In 1990, the ﬁrst year of this study, ZT
signiﬁcantly decreased percent defoliation due to early blight (Figure 2 & 3) as
compared to a conventional tillage system. The mechanism may in part be due to the
near-complete covering of the soil surface and a reduction in fungal spore dispersal.
Alternatively, the primary effect may be indirect through altered host productivity and

reaction to infection (Sumner et al. 1986). For example, delayed senescence is known

 

 

 

to be :
Heube
bioma
plants

andfr

under
signlf

3881111

inocu
above
persis
inocu
PIOdt
plots.
Curve
mean
the d
Obser
funct
ledtx
Phen.

dfifol

 

 

108
to be associated with decreased susceptibility to defoliation due to BB (Horsfall and
Heuberger 1942; Jones and Jones 1986; Thomas 1948). Zone tillage enhances root
biomass accumulation and soil exploration (Grajauskis 1990) which could result in
plants with more vigor. Visually, plants appeared greener longer throughout the season
and fruit maturity was delayed (Chapter 3) as compared to CT.

Transformation of percent defoliation with the logistic model provided some
understanding as to the impact of zone tillage. In 1990, the level of the curve was
signiﬁcantly lower in ZT plots as compared to CT plots (Figure 2 and 3). Based on the
assumption that initial levels of natural inoculum were randomly distributed in ZT and
CT plots prior to planting any tomatoes, ZT may have reduced the potential of initial
inoculum to disperse or may have delayed senescence in host plants as described
above, and the onset of an epidemic. In 1991, tomato debris from the 1990 crop
persisted on the soil surface in ZT plots and functioned as a ready source of initial
inoculum. The high incidence of collar rot, an uncommon disease in the northem
production regions, attests to the inoculum levels that must have been present in ZT
plots. Data transformed with the logistic model (inset Figure 2 and 3) illustrate the ZT
curves were substantially higher than CT curves. However, in both 1990 and 1991, the
mean rate of the epidemic (r) was lower in ZT plots compared to CT plots, although
the differences were not always signiﬁcant. The logistic data and crop productivity
observations suggest ZT impacts components of the host-patho gen interaction in a
functional manner similar to rate-reducing resistance (Kuhn et a1. 1978), only the rate-
reducing resistance is not mediated by host genotype, but by cultural impacts on
phenotype. Additional work is required before the mechanism of reduced EB

defoliation will be adequately known.

 

by till:
mold :

declin

fungaf
Integr
and it
must
resen

Papai

1992
rotati

simil

clear
(Cha
00ml
DSV

(Fig

redo

appg

 

 

109

The proportion of total fruit mold caused by EB, ANTH or SR was not affected
by tillage in 1990 and 1991 (Table 11) even though the absolute amount of fruit with
mold symptoms changed. There was a slight tendency for the incidence of ANTH to
decline in ZT plots and the incidence of SR to increase, but these changes were not
dramatic. These data suggest ZT uniformly impacted each component of the foliar-fruit
fungal complex. This is important when devising integrated management systems.
Integrated disease management systems are designed to control a complex of problems
and if the proportion of each problem changes, the dynamics of disease management
must also be adjusted and so, become more complex. We feared that ZT may provide a
reservoir base of crop residue for the saprophytic persistence of R solani (Neate 1987;
Papavizas 1970) resulting in proportionally more SR problems in ZT plots compared to
CI‘ plots. This was not the case except in 1992 in continuous tomato ZT plots. Also in
1992, ZT combined with rotation had proportionally less SR than CT combined with
rotation, although the total amount of disease was higher. Cultural practice also had
similar effects in both FMT and PRT systems.

The reason for the higher level of disease in 1992 in ZT x rotation plots is not
clear although the total weight of marketable fruit was also substantially higher
(Chapter 3). Plots not sprayed appeared to be affected most dramatically by the ZT x R
combination (Figure 7). In contrast, full rate fungicide applied weekly or after every 25
DSVs provided control of fruit mold comparable to the CT x rotation combination
(Figure 7). "

In 1991, CT proved to be important for reducing initial inoculum and, overall,

reduced incidence of disease as compared to ZT plots. In the absence of rotation, Cl‘

appears to be essential to bury crop debris and reduce overwintered inoculum levels. A

 

 

 

2 to 3 yea
(Horsfall t
commerci:
the impac
inoculum
A]
accumula
as a stant
liberal sc
Premise 1
program
1992, a l
nation
1
cases as
there w;
(Figure
mold in
AUDPr
Plots m
aPPoni
with 5]
appear:
broom,

 

 

110

2 to 3 year rotation to non-solanaceous crops is a standard recommendation for tomato
(Horsfall & Heuberger 1942, Sherf and MacNab 1986; Jones et al. 1991). Most
commercial growers would not grow continuous tomato. Our purpose was to compare
the impact of continuous tomato on disease level and to provide challenging levels of
inoculum to test the TOMCAST model.

Applications of chlorothalonil according to the TOMCAST model after the
accumulation of every 20 DSVs is considered a liberal schedule and is not as effective
as a standard spray schedule in Iowa (Gleason et al. 1992). In 1990, we chose the
liberal schedule of 20 and 25 DSVs as thresholds for fungicide application on the
premise natural levels of inoculum would be low. In 1991, a more conservative spray
program was adopted to account for predicted high levels of overwintered inoculum. In
1992, a DSV 25 threshold treatment was again included to determine the contribution
rotation and tillage may offer in a reduced—sprays program.

TOM-CAST based spray schedules did not provide control of defoliation in all
cases as compared to plots sprayed weekly (Figure 2 and 3, Table 5,7 and 9). However,
there was no consistent relationship between AUDPC and incidence of SR or ANTH
(Figure 8). No relationship was observed in PRT. In 1992, incidence of ANTH fruit
mold in the FMT production system increased 0.2% for each 100 percent—days of
AUDPC (non-transformed AUDPC). However, main effect mean AUDPC values of
plots treated with fungicide did not exceed 1000. In 1992, 1000 percent days was
approximately correlated with 60% ﬁnal percent defoliation. Likewise, percent fruit
with EB symptoms increased 0.3% to 2.3 % for each 100 percent-days. Therefore it
appears a certain level of defoliation can be tolerated before incidence of fruit mold

becomes a problem.

 

 

For t
scheduled cl
weekly app]
in 1990, 19‘
rate spray s
rotation in
fungicide a

An
of decrease
application
managed l
rates at a]
way treat
and this w
solani. Tl
ATHR an
intervals.
SR but tl

C
“1mm et
believed
iﬂoculun
incidenc

levels 01

 

 

111

For example, with the exception of FMT in 1990, selective TOM-CAST
scheduled chlorothalonil applications reduced percent fruit with mold comparable to a
weeldy application schedule (Figure 5 to 7). In 1991 and 1992 in the case of FMT, and
in 1990, 1991, and 1992 in the case of PRT, the most liberal TOMCAST-based, full-
rate spray schedule provided control of fruit mold when combined with ZT, CT and
rotation in 1990, 1991, and 1992, respectively. This represented 64% to 80% fewer
fungicide applications.

Application of reduced fungicide rate (2.8 L ha") introduced more risk in terms
of decreased fruit quality, as compared to full rate (4.2 L ha") treatments, even if
applications were more frequent. Therefore, reduced total fungicide input appears to be
managed best by proper scheduling of fewer sprays as opposed to reduced frmgicide
rates at a more frequent schedule. Weekly frmgicide sprays, as compared to the no
spray treatment, tended to decrease the proportion of fruit mold caused by C. coccodes
and this was complemented by an increase in the proportion of SR, caused by R.
solani. These data suggest chlorothalonil is not equally effective in the control of
ATHR and SR. McCarter and Barksdale (1977) noted fungicide applications at regular
intervals during fruit development can be moderately effective in providing control of
SR but this is not considered a reliable, economic approach (Jones et al. 1991).

C. coccodes is able to colonize and sporulate on senescent foliage (Farmer 1959;
Illman et a1 1959; Pantidou and Schoeder 1955; Younkin et al. 1944) and this is
believed to act as a ”bridge” between initial inoculum from the soil and secondary
inoculum that affects fruit. In this study, the poor relationship between AUDPC and

incidence of ANTH fruit rot suggests this is not the mechanism by which inoculum

levels of C. coccodes build up. Alternatively, defoliation may affect the

 

 

Moon
1986). (
sources
seconds

infectio

1990 P
may at
fruit It
percen
solani
borne

torna .

proce
early
half I
load.
(Ban
OPPC
Wag
tthl
leve

incr

 

F—

112

microenvironment or fruit susceptibility and favor ANTH fruit rot (Sherf and MacNab
1986). Considerable research is required to determine the relationship between initial
sources of inoculum, the relative importance of foliar infection, the potential of
secondary buildup of inoculum on leaf tissue, and the inoculum responsible for fruit
infection.
The correlation between incidence of EB and ANTH, with the exception of
1990 PRT data (Table 16), suggests conditions that favor EB also favor ANTH. This
may account for the effectiveness of the TOM CA ST model to provide control of both
fruit molds. There was no consistent correlation between AUDPC value, percent EB or
percent ANTH with SR. This was foreseen since the biology and epidemiology of R
solani differs considerably as compared to A. solani and C. coccodes. Rsolani is a soil
borne pathogen and does not produce spores. The pathogen is a serious concern with
tomato fruit in contact with soil (Jones and McCarter 1974).
Genetic and a gronornic production differences between fresh market (FMT) and
processing tomato (PRT) production systems also impacted the level and proportion of
early blight (EB), anthracnose (ANTH), and soil rot (SR). FMT plants were planted at

half the density as compared to PRT plants and bore a heavier early and total fruit

 

load. Earliness and fruit load are associated with increased susceptibility to A. solani
(Barratt and Richards 1944; Horsfall and Heuberger 1942; Sherf and MacNab 1986) as
opposed to a true resistance mechanism. Maximum percent defoliation of FMT plants
was two-fold greater in 1990 as compared to PRT plants and continued to be higher
throughout the study, even with a PRT cultivar change in 1991. Presumably, inoculum
levels associated with overwintered debris, volunteer plants, or alternative hosts

increased from one year to the next leading to progressively higher levels of defoliation

 

 

 

each year
influence
Fr

This may
in 1990.
(Summer
in the bi
demonst
precedes
reported

to sugar

popular

ﬁelds 1:

Study, :

the 1111]

Picked
PRT f.
machl
1948),
1992)
0f tot

andﬁ

Table

 

F—

113

each year. However, the dramatic increase in percent defoliation in 1992 was
inﬂuenced by late harvest dates and a prolonged period for epidemics to develop.
Fresh market tomato are thin skinned compared to processing tomato cultivars.
This may account for the high incidence of SR on FMT fruit as compared to PRT fruit
in 1990. R. solani is inﬂuenced more by the previous crop than by tillage system
(Sumner et al. 1986; Rush and Winter 1990) although there is considerable variability
in the biology and ecology among anastomosis groups. Hayslip and Stall (1959)
demonstrated SR is much more severe when a grass plus a white clover cover crop
precedes tomato than when grass alone precedes tomato. Rush and Winter (1990)
reported sugar beets following alfalfa had the highest incidence of disease as compared
to sugar beets following cotton, fallow or sunﬂower. Brunson et al. (1993) noted
populations of R. solani in a cantaloupe production system were signiﬁcantly higher in
ﬁelds previously planted to crimson clover and subterranean clover cover crops. In our
study, a rye crop preceded the tomato crop. Limited information is available concerning
the impact of tillage and previous crop on incidence of soil rot in tomato fruit.
FMT and PRT production systems differ in the mode of harvest. FMT fruit are

picked in multiple harvests at the breaker stage or riper, and marketed. Harvest of ripe

 

PRT fruit is often delayed for extended periods to allow optimum timing for oncebver
machine harvesting. Because ANTH symptoms appear only as fruit ripen (Fulton
1948), incidence of ANTH at harvest can be high in PRT production systems (Precheur
1992) but not FMT systems. In our study, ANTH comprised approximately 22 - 32%
of total PRT fruit mold as compared to 7.0 - 11% of total FMT fruit mold (Figure 5
and 6). However, ANTH can be a post-harvest problem if consumption is delayed.

Table 14 shows that TOMCAST based sprays can be equally effective as weekly

 

 

 

sprays to E-
Fin
relationshi
compared
primarily
or pedicle
stern tissr
In
terms of
season C
3, Apper
productir
producti
and che:
required
si'stem
inputs t
Ultimar
and pe;
the fat
Hower
krrowl.
Whit

SlStcn

 

1 14
sprays to limit latent infections and ensure marketability.

Finally, because FMT fruit was removed from vines weekly, there is less of a
relationship between the AUDPC values and incidence of EB fruit mold (Figure 8) as
compared to PRT. Although A. solani can infect fruit directly, fruit invasion occurs
primarily through the stem end (Horsfall and Heuberger 1942) apparently via the calyx
or pedicle. Premature removal of fruit likely limits migration of the pathogen from
stem tissue into the fruit.

In this study, in addition to the affect on disease levels, substantial beneﬁt in
terms of reduced wind and water soil erosion, wind damage to plants, reduced early
season Colorado potato beetle populations, and yield were achieved with ZT (Chapter
3, Appendix B). This cultural practice holds considerable promise for vegetable-based
production systems. A reduced-sprays program was effectively incorporated into the
production system and was successful, in part, due to the integrative impacts of cultural
and chemical based control strategies. However, a higher level of management was
required when both strategies were combined. This combination makes the production
system more dependent on an enhanced knowledge base as opposed to production
inputs (Fretz et al. 1993). This will prove to be a challenge for the farm manager.
Ultimately, integrated management of indigenous diseases (i.e. diseases that originate
and persist at the farm level) such as EB, ANTH and SR is dependent on the ability of
the farm manager to integrate available knowledge into a working production system.
However, private and public research and extension must provide a framework and
knowledge resource base from which the farm manager can draw. Currently, in tomato

production systems, the knowledge base lags behind the stimulus to change production

SYSICIIIS.

 

 

 

 

Abdul-Bald, r
utilizing hairy

Barratt, R.W.
Lycopersicon

Batson, W.,Jr
57:453-456.

Brunson, Kl
influence yie
production. 1

Campbell C.
John Wiley .

Coolrnan, R.
environment

Doss, B.D,,
cover crop r

Farmer, ],V
atramentarir

 

 

 

l 15
LITERATURE CITED

Abdul-Bald, A.A., and J .R. Teasdale. 1993. A no-tillage tomato production system
utilizing hairy vetch and subterranean clover mulches. HortScience: in press.

Barratt, R.W., and M.C. Richards. 1944. Altemaria blight versus the genus,
Lycopersicon. N.H. Agric. Exp. Stn. Bul. 82. 25 pp.

Batson, W.,Jr. 1973. Characterization and control of tomato fruit rot. Plant Dis. Rep.
57:453-456.

Brunson, K.E., S.C. Phatak, J.D. Gay, and DR. Sumner. 1993. Winter cover crops
inﬂuence yields, nematodes, and soil-bome pathogens in sustainable cantaloupe
production. HortScience 28:453 (abstr.).

Campbell CL. and L.V. Madden. 1990. Introduction to plant disease epidemiology.
John Wiley & Sons, New York.

Coolman, R.M., and GD. Hoyt. 1993. The effects of reduced tillage on the soil
environment. HortTechnology 3: 143-145.

Doss, B.D., J.L. Turner, and CE. Evans. 1981. Inﬂuence of tillage, nitrogen, and rye
cover crop on growth and yield of tomatoes. J. Amer. Soc. Hort. Sci. 106:95-97.

Farmer, J .V. 1959. Infection studies on tomato anthracnose caused by Colletotrichum
atramentarium (B & BR) Taub. MS Thesis, The Univ. of Western Ontario.

Fretz, T.A., D.R. Keeney, and SB. Sterrett. 1993. Sustainability: Deﬁning the new
paradigm. HortTechnology 3:118-126.

Frye, W.W., and R.L. Blevins. 1989. Economically sustainable crop production with
legume cover crops and conservation tillage. J. Soil Water Conserv. 44:57-60.

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

Gleason, M.L., S.E. Taylor, H.G. Taber, and ML. Hockmuth. 1992. TOM-CAST on
tomatoes in Iowazeffect of model threshold and sensor location on control of fungal
diseases. Phytopathology 82:1164 (abstr).

Grajauskis, J .J . 1990. Effects of nitrogen, rye cover and zone tillage on the yield of
fresh market tomatoes in three tillage systems. MS Thesis. Michigan State University.

70 pp.

Hayslip, N .C. and RE. Stall. 1959. Severity of soil-rot caused by Rhizoctom’a solani
as inﬂuenced by former pasture crop. Plant Dis. Rep. 43:818-820.

 

 

Horsfall, J .(
tomatoes. C

ﬂlman Wl
Ontario. Ca

Jones, J.P.,
mixtures ar

Jones, C.W
cultural am

Jones, J.B.
Diseases. J

Kelly, W.(
HortScienc

Knavel, D
performan.
Sci. 102:31

Kuhn R.C
against tw

Little, up
John Wile

Madden, j
for Altern

MCCarter
Controllin
Rep. our

MCKBOW
Yield, dis

Morse, R

“are. 8.
Wheat. T

Pantidou
for toma

 

 

 

116

Horsfall, J .G., and J .W. Heuberger. 1942. Causes, effects and control of defoliation on
tomatoes. Conn. Agr. Exp. St Bull. 456.

lllrnan W.I., R.A. Ludwig, and J. Farmer. 1959. Anthracnose of canning tomatoes in
Ontario. Can. J. Bot. 37:1237-1246.

Jones, J .P., and J.B. Jones. 1986. Control of early blight of tomato with foliar spray
mixtures and high fertilizer rates. Proc. Fla. State Hort. Soc. 99:318-321.

Jones, C.W., and SM. McCarter. 1974. Etiology of tomato fruit rots and evaluation of
cultural and chemical treatments for their control. Phytopathology 64:1204—1208.

Jones, J.B., J.P. Jones, R.E. Stall, and T.A. Zitter. 1991. Compendium of Tomato
Diseases. APS Press, St. Paul,MN. 73pp.

Kelly, W.C. 1990. Minimum use of synthetic fertilizers in vegetable production.
HortScience 25:168-169.

Knavel, D.E., J. Ellis, and J. Morrison 1977. The effects of tillage systems on the
performance and elemental absorption by selected vegetable crops. J. Amer. Soc. Hort.
Sci. 102:323-327.

Kuhn R.G., .W. Ohm, and GE. Shaner. 1978. Slow leaf-rusting resistance in wheat
against twenty two isolates of Puccinia recondita. Phytopathology 68:651-656.

Little, T.M., and F.J. Hills. 1978. Agricultural Experimentation: Design and Analysis.
John Wiley and Sons, New York, p.350.

 

Madden, L.V., S.P. Pennypacker, and AA. MacNab. 1978. FAST, a forecast system
for Altemaria solani on tomato. PhytOpathology 68:1354-1358.

McCarter, S.M., and TH. Barksdale. 1977 . Effectiveness of selected chemicals in
controlling Rhizoctonia fruit rot of tomato in greenhouse and ﬁeld tests. Plant Dis.
Rep. 61:811-815.

McKeown, A.W., R.F. Cerkauskas, and J .W. Potter. 1988. Inﬂuence of strip tillage on
yield, diseases and nematodes of tomatoes. J. Amer. Soc. Hort. Sci. 113:328-331.

Morse, RD. 1993. Components of sustainable production systems for vegetables-
conservation of soil moisture. HortTechnology 3:211-214. ~

Neate, SM. 1987. Plant debris in soil as a source of inoculum of Rhizoctonia solani in
wheat. Trans. Br. Mycol. Soc. 88:157-162.

Pantidou, M.B., and W.T. Schroeder. 1955. Foliage as a secondary source of inoculum
for tomato anthracnose. Phytopathology 45 :33 8—345 .

 

Papavizas, G.C
108-122 in: Pa
of California P

Phatak, S.C. ll
27:738-741.

Phillips, R.E.,
till agriculture.

Pitblado, RE.
ﬁeld tomatoes

Pitblado, RE.
and Food. Put

Precheur, R.P.
Management .

Price, H.C,, a
till system A

Pram, are
HortSicence 1

Rush, QM” ,
and crown to

Sarrantonio, j
crops in veg:

Sharlot, (3,, a

e’ll’TCSSitln o}
1186.

Shelby, P.P.,
PTOduotion a

Hort. Sci. 11

Sheena. c.
Vegetable pr.

Sheft. An,
edition, Johr

Stephens, C
HORSCieRCe

 

117

Papavizas, G.C. 1970. Colonization and growth of Rhizoctonia solani in soil. Pages
108-122 in: Parmeter, J.R.,Jr., (ed.), Rhizoctonia Solani, Biology and Pathology. Univ.
of California Press. 255 pp.

Phatak, S.C. 1992. An integrated sustainable vegetable production system. HortScience
27:738-741.

Phillips, R.E., R.L. Blevins, G.W. Thomas, W.W. Frye, and SH. Phillips. 1980. No-
till agriculture. Science 208:1108-1113.

Pitblado, RE. 1988. Developement of a weather-timed fungicide spray program for
ﬁeld tomatoes. Can. J. Plant Path. 10:371 (abstr.).

Pitblado, RE. 1992. Development and implementation of TOM-CAST. Ont. Min. Agr.
and Food. Publ. p.18.

Precheur, R.P., R.R. Riedel, M.B. Bennett, K.L. Wiese, and J. Dudek. 1992.
Management of fungicide residues on processing tomatoes. Plant Dis. 76:700-702.

Price, HQ, and RA. Baughan. 1987. Establishment of fresh market tomatoes in a no-
till system. Acta Hort. 198:261-268.

Putnam, AR. 1990. Vegetable weed control with minimum herbicide inputs.
HortSicence 25:155-159.

Rush, C.M., and S.R. Winter. 1990. Inﬂuence of previous crop on Rhizoctonia root
and crown rot of sugar beet. Plant Dis. 74:421-425.

Sarrantonio, M. 1992. Opportunities and challenges for the inclusion of soil-improving
crops in vegetable production systems. HortScience 27:754—758.

Shaner, G., and RE. Finney. 1977. The effect of nitrogen fertilization on the

expression of slow-mildewing resistance in Knox wheat. Phytopathology 70:1183-
1186.

Shelby, P.P.,Jr., D. L. Coffey, N. Rhodes, Jr., and LS. Jeffery. 1988. Tomato
production and weed control in no-tillage versus conventional tillage. J. Amer. Soc.
Hort. Sci. 113:675-678.

Sherman, C. 1992. Cover crops, nitrogen cycling, and soil properties in semi-irrigated
vegetable production systems. HortScience 27:749—754.

Sherf, A.F., and A.A. MacNab. 1986. Vegetable diseases and their control. 2nd
edition, John Wiley & Sons, NY. 728 pp.

Stephens, C.T. 1990. Minimizing pesticide use in a vegetable management system.
HortScience 252164-168.

 

 

Sunmer, D.
Conservatic

Thomas, H
tomatoes tc

Wallace, R
successful

Wein, H.C
synthetic ft

Wilson, J .l
to anthract

Younkin, :
Lycopersic

 

 

118

Summer, D.R., ED. Threadgill, D.A. Smittle, S.C. Phatak, and A.W. Johnson. 1986.
Conservation tillage and vegetable diseases. Plant Dis. 70:906-911.

Thomas, HR. 1948. Effect of nitrogen, phosporous, and potassium on susceptibility of
tomatoes to Altemaria solani. J. Agric. Res. 76:289-306.

Wallace, R.W., and RR. Bellinder. 1992. Alternative tillage and herbicide options for
successful weed control in vegetables. HortScience 27:745-749.

Wein, H.C. 1990. Sustainable commercial vegetable production with minimal use of
synthetic fertilizers and pesticides: a postlude. HortScience 25:170-171.

Wilson, J .D., and HA. Runnels. 1949. Comparitive susceptibility of tomato varieties
to anthracnose and their response to spraying. Ohio Agr. Exp. St. Bull. 685.

Younkin, S.G., NJ. Riverton, and A.W. Dimock. 1944. Foliage infection of
Lycopersicum esculentum by Colletotrichum phomoides. Phytopathology 34:976-977.

 

 

 

CONSERVA

 

 

CHAPTER III

 

CONSERVATION TILLAGE AND ROTATION TO CUCUMBER IN TOMATO
PRODUCTION SYSTEMS.

119

 

 

 

 

Zone ti?
(FMT) and pr:
cucumber, use
input. A multi
fall-seeded rye
centers, the sc
no soil inversf
throughout tin
utilized spring
less than 4%.
trait and mi;
We was seed
tomato was I
sigll‘lflcantly
plots were so
coIntentional
(1992) and l
inhence) tot
(TOMCAST
yield in the

80% fCWer :

 

 

120

ABSTRACT

Zone tillage (ZT) was compared to conventional tillage (CT) in a fresh market
(FMT) and processing tomato (PRT) production system that included rotation to
cucumber, use of rye cover crops, a mustard green manure, and reduced fungicide
input. A multi-factorial experimental design was used from 1990-1992. For ZT, after
fall-seeded rye was chemically desiccated in Apr in strips 0.46 m wide on 1.5-m
centers, the soil was fractured to a depth of 35 cm with minor surface disturbance and
no soil inversion. Remaining inter-row rye was desiccated in May and persisted
throughout the summer providing up to 90% or more cover of the soil surface. CT
utilized spring mold board plowing and discing. Soil surface residue in CT plots was
less than 4%. ZT enhanced PRT yield in 1990, did not affect FMT marketable NO.1
fruit and weight of cull fruit and decreased weight of FMT No.2 fruit. After harvest,
rye was seeded followed by C1“ and ZT in the spring and cucumber (pickling type) or
tomato was planted. Cucumber yield from a once-over harvest (Jul 25) was not
signiﬁcantly affected by tillage. After the cucumber harvest, CT was performed and
plots were seeded to a mustard crop as a late summer fallow. All plots were
conventionally tilled mid-Sep, seeded to rye, followed by ZT or CT in the spring
(1992) and planted to tomato. Rotation did not affect FMT nor PRT yield in 1992. ZT
enhanced total yield of PRT (P=0.05) and did not irnpact yield of FMT. Forecast
(TOMCAST) generated chlorothalonil spray schedules did not compromise marketable
yield in the FMT or PRT production system during the 3 yr study but required 45 % to

80% fewer sprays as compared to a standard weekly spray program.

 

 

 

 

 

 

 

Conservation

equal success in veg

compared to ﬁeld or

 

Vegetable productio

cropping of high val

, Specialized producti
overhead costs, imp
sequences that woul
enterprises are able
1992) despite prove
the soils (Frye and
people and demand
importance of pestic
production systems 1

Advancemer
in numerous vegeta'
1994; Doss et al. 1!
1988; Shelby et al.

, marketable yield of

; biennial crop cycle

green manure. Zon

Plants are to grow

 

 

 

121

INTRODUCTION

Conservation tillage and cover cropping systems have not been adopted with

equal success in vegetable production systems (Kelly 1990; Sarrantonio 1992) as

 

compared to ﬁeld crop production systems (Gebhardt et al. 1985; Phillips et al. 1980).
Vegetable production systems often have a high value land base with multiple

cropping of high value crops, using intensive tillage and fertilizer inputs (Kelly 1990).

 

Specialized production systems, often with substantial investments in mechanized
overhead costs, impact a growers ability or desire to include alternative cropping
sequences that would enhance or build up soils. For example, few vegetable

enterprises are able to include long rotations to a legume cover crop (Sarrantonio

1992) despite proven beneﬁts toward the long term sustainability and productivity of

 

the soils (Frye and Blevins 1989). Vegetable crops are also directly consumed by

people and demand for quality is high, directly impacting the perceived and real

 

importance of pesticide inputs. Considerable research is required to make vegetable
production systems more sustainable and less input intense.

Advancements in reduced tillage and cover crop use have recently been made
in numerous vegetable production systems including tomato (Abdul~Baki and Teasdale
1994; Doss et al. 1981; Knavel et al. 1977; McKeown et al. 1988; Price and Baughan
1988; Shelby et al. 1988). The purpose of this study was to determine the impact on
marketable yield of fresh market and processing tomato using zone tillage within a
biennial crop cycle (tomato/cucumber) that included rye cover crops and a mustard

green manure. Zone tillage (ZT) is deﬁned as the fracturing of the soil below where

 

Plants are to grow (Grajauskis 1990).

 

 

 

 

Location and design

have been described
growth, and dete ‘
plot of 3 fungicide tr
not sprayed and plot

every 20 (1990) or 1

Assessment of surf
was estimated accor
monthly intervals in
cm apart was laid 1
operation) across 3 ‘
determined by the r

knots multiplied by

Assessment of plan
were obtained (as <
each season. Plant
second, fifth and e
and ﬁfteenth plant

averages of 3 plan

recorded in 1990.

 

 

 

 

 

122

MATERIALS AND METHODS

Location and design of ﬁeld experiments. Location, design and cultural practices
have been described previously (Chapter 2). Assessments of plant phenology and
growth, and determination of percent surface rye residue was done in each sub~sub-
plot of 3 fungicide treatments. The 3 sub-sub-plots were plots sprayed weekly, plots
not sprayed and plots sprayed with full rate chlorothalonil after the accumulation of

every 20 (1990) or 15 (1991, 1992) disease severity values.

Assessment of surface rye residue. Percent rye residue coverage of the soil surface
was estimated according to the method of Slonneker and Moldenhauer (1977) at
monthly intervals in 1991 and 1992. A 1.3 cm diameter nylon rope with 50 knots 15.2
cm apart was laid lengthwise in plots or diagonally (i.e. not parallel to tillage
operation) across 3 plot rows and 3 alleys, inclusive. Percent surface residue was

determined by the number of knots that touched residue divided by the total number of

knots multiplied by 100.

Assessment of plant phenology and growth. A 6 m row length from which all data
were obtained (as described previously in Chapter 2), was delineated by ﬂags early in
each season. Plant height and phenology data in 1991 and 1992 were recorded for the
second, ﬁfth and eighth plant from the south ﬂag in FMT rows, and the ﬁfth, tenth
and ﬁfteenth plant from the south ﬂag in PRT rows. Data were analyzed on the

averages of 3 plants per plot per tomato type. Plant phenology and growth were not

recorded in 1990.

 

 

 

 

 

 

 

 

Tomato harvest and

multiple harvested ((1

reached the breaker

Nol’s and non mark
diseases and reporte
Processing to
1992) were treated
shown in Table 1).
Subsequently, two
of people according
of anthracnose, earl:

elsewhere (Chapter

Cucumber harvest
over harvested Jul 1

sorted for quality. 1

 

 

 

 

 

.123

 

Tomato harvest and evaluation. Fresh market tomato (FMT) cv ’Pik -Rite’ were
multiple harvested (dates shown in Table 1) from a 6 m row section when fruit
reached the breaker stage or riper. All fruit were graded twice, once using market
standards and again for disease symptoms. Fruit were graded for size on a commercial
grader. Sizes included large fruit (No. 1) with a diameter >67 mm and medium (No.
1) fruit with a diameter of 54-67 mm. Marketable fruit with blemishes were labelled
No.2’s and non marketable fruit was culled. Fruit were also sorted for symptoms of
diseases and reported elsewhere (Chapter 2, Appendix B).

Processing tomato (PRT) cv. OHIO 7870 (1990) and HEINZ 8704 (1991 &
1992) were treated with Ethrel and harvested by a once-over harvest (rates and dates
shown in Table 1). Fruit from the 6 m harvested area was weighed and pooled.
Subsequently, two subsamples collected in 20 L pails were rated by independent teams
of people according to market standards for ripe, green and cull fruit or for incidence
of anthracnose, early blight and soil rot. Data on incidence of disease are reported

elsewhere (Chapter 2)-

Cucumber harvest and evaluation: Cucumber cv. Flurry (pickling type) were once-
over harvested Jul 25 and graded on a commercial grader for size and subsequently

sorted for quality. Grade size and quality classes are deﬁned in Table 7.

 

 

 

 

    

 

TABLE 1: Date of .
number of fungicide .

 

'lulian Day of Year
bDate of intitial applic
“Fungicide applied all

‘1. - low rate of Br.“
' Truman not applit
'lniﬁal spray was inr

 

124

TABLE 1: Date of harvest, date and rate of ethrel treatment, fungicide treatment,
number of fungicide applications, and date of initial fungicide application in years
1990 to 1992.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ACTIVITY OR YEAR
TREATMENT
1990 1991 1992
HARVEST DATES Aug 7(218)‘ Jul 30(210) Aug 19(231)
OF FRESH MARKET
TOMATO Aug 15(226) Aug 5(216) Aug 25(237)
Aug 22(233) Aug 12(223) Sep 1(244)
Aug 28(239) Aug 21(232) Sep 9(252)
Sep 5(247) Aug 27(238) Sep 15(258)
Sep 12(254) Sep 22(265)
HARVEST DATE Sep 18(260) Aug 29(240) Sep 29(273)
OF PROCESSING
TOMATO
DATE OF ETHREL Sep 5 Aug 16 Sep 14
APPLN
RATE OF ETHREL 2.8 L ha" 4.2 L ha'l 4.2 L ha‘l
APPLIED
FUNGICIDE TREATMENT NUMBER OF FUNGICIDE APPLICATIONS
WEEKLY 15(Jun 15)” 11 (Jun 16) 13 (Jun 25)
DSVc 15L‘I NAe 6 (JUN 26)” 5 (Jul 16)”
DSV 15H NA 6 (Jun 26) 4f
DSV 20L 4 (Jul 11)b 4 (Jun 26) 4 (Jul 16)
4 (Jul 11) 4 (Jun 26) 3f
| DSV 25L 3 (Jul 11) NA NA
| DSV 25H 3 (Jul 11) NA 3 (Jul 16)
l NO SPRAY 0 0 0

 

 

 

 

 

‘ Julian Day of Year
” Date of intitial applimtion for weekly or TOMCAST-based spray programs
° Fungicide applied after the accumulation of every 15, 20 or 25 disease severity values
‘ L = low rate of Bravo 720 (2.8 1 ha"), H = high rate of Bravo 720 (4.2 L ha“)
° Treatment not applied during this year

'lnitial spray was inadvertantly omitted. First application = Jul 30

 

 

 

Data analysis. All dz
(Little and Hills 1972
Programming Enterpz
lansing, MI). Only i
as a split plot of tilla
way factorial analysi
using 8 (n=112) and
to determine the effe
ANOVA was used 1
for the main factors
Sitilliflcant, signiﬁer
by Planted F tests a
enor term Means t
appropriate LSDs i
mean s‘lllare error .
aﬁeld tile that Ina]
in1990, In 1991, r
subroutine of MS'I
PersiSted and iIIIpa
in CT SI1b~plots in
complete “when

 

125

Data analysis. All data had homogenous variance as determined by Bartlett’s test
(Little and Hills 1978) and analysis of variance was performed with Plot-IT (Scientiﬁc
Programming Enterprises, Haslett, MD or MSTAT-C (Michigan State University, E.
Lansing, MI). Only in 1992 was a full three way factorial model used with fungicide
as a split plot of tillage and tillage as a split plot of rotation. In 1990 and 1991 a two
way factorial analysis was performed with fungicide treatment as a split-plot of tillage
using 8 (n=112) and 4 (n=56) replications, respectively. The experiment was designed
to determine the effect of rotation, tillage or fungicide treatment and their interactions.
ANOVA was used to partition the degrees of freedom and associated sums of squares
for the main factors and their associated interactions. If the interactive effects were not
signiﬁcant, signiﬁcant effects due to rotation or tillage (main effects) were determined
by planned F tests calculated from the analysis of variance table using the appropriate
error term. Means from signiﬁcant fungicide treatment effects were separated with
appropriate LSDs based on a signiﬁcant F value, calculated using the overall residual
mean square error of the ANOVA table (i.e. protected LSD). Certain plots were above
a ﬁeld tile that malfunctioned during the experiment. No values were obvious outliers
in 1990. In 1991, values for 4 cucumber plots were estimated using the MISVALEST
subroutine of MSTAT-C. No tomato plots were affected in 1991. In 1992, the problem
persisted and impacted 2 replications of a complete treatment (i.e. many sub-sub—plots
in CT sub-plots in rotation main plots). With unreliable data for 2 replications of

complete treatments, 1992 data were analyzed over the remaining 2 replications only

(n=56 rather than 112).

 

 

 

 

The ZT syster
Management decisior
strips and ﬁnal desic
emergent herbicides.
system, and not dire

Desiccated r)
season. Data for 199
Surface residue in C
plots. Percent residu
residue measured 1e
low level of residue

the season, this area

TABLE 2: Percent

Turner.

 

 

 

 

 

126

RESULTS

The ZT system required a higher level of management than the CT system.
Management decisions included timing of herbicide application for preparation of
strips and ﬁnal desiccation of rye, timing of zone tillage and precision timing of post-
emergent herbicides. However, multiple advantages associated with the ZT production
system, and not directly related to plant productivity, were observed.

Desiccated rye in ZT plots progressively lodged and persisted throughout the
season. Data for 1992, similar to data of 1991 (not shown), is presented in Table 2.
Surface residue in CT plots was 3.3% or less as compared to greater than 90% in ZT
plots. Percent residue measured across the plot in ZT plots was lower than percent
residue measured lengthwise between 2 rows. Early in the season, row centers had a

low level of residue due to the strip application of herbicide and zone tillage. Later in

the season, this area was covered by tomato foliage and not assessed.

TABLE 2: Percent surface rye residue Q standard error) in plots conventionally tilled
(CT) or managed by a zone tillage (ZT) system in 1992.

 

 

 

 

 

 

 

 

 

TILLAGE JUN 25 JUL 23 SEP 3
CW1 Lwl cw Lw Lw
CT 2.5 1 0.5 3.3 1 0.6 2.9 1 0.6 1.1 1 0.3 1.6 1 0.5
ZT 90.0 1 0.9 94.4 1 4.2 90.4 1 1.0 96.9 1 1.2 93.6 1 1.9

 

 

 

1 Percent residue was determined cross-wise (CW) across 3 row centers and alleys intemal
to each plot.

2 Percent residue was determined length-wise (LW) between two rows within @3011 plot.

 

Soil maintena
several rain storms 0
compared to ZT plot
plants from excessiv
the tomato plants. A
to 100% of tomato 1
surface in CI‘ plots.
persistence of surfac
damage to plants.
1990 SUMMER SI
revealed that tillage
culls of fresh marke
4A). Total yields w
0“ the total yields (

Fungicide a.
fruit in plots treater
DSVS was Similar 1
We Weekly, Dsr
was compromised :
regard to Weight 01
(Table 3). Wei ght

and DSV 25H trea
WW to or p‘

culls. Total yield 1

 

 

127

Soil maintenance qualities of the rye residue was qualitatively observed after
several rain storms over the 3 year study. Soil erosion was obvious in CT plots as
compared to ZT plots. The potential of reducing wind erosion of soil and protecting
plants from excessive winds was observed on Jul 2, 1992, 3 weeks after ﬁeld setting
the tomato plants. A severe sand storm occurred and immediately after the storm, up
to 100% of tomato plants were noted to be wind wiped and tilted toward the soil
surface in CT plots. Plants in ZT plots appeared to remain upright. ZT allowed for the
persistence of surface rye residue to limit soil and wind erosion, and wind-wiping
damage to plants.

1990 SUMMER SEASON - Tomato fruit yield and quality: Analysis of variance
revealed that tillage did not affect yield of No. 1 large fruit, No.1 medium fruit, nor
culls of fresh market tomato but weight of No.2 fruit was less in ZT plots (Table 3,
4A). Total yields were not affected by tillage but ZT appeared to delay maturity based
on the total yields obtained on each harvest date (Figure 1).

Fungicide affected yield and fruit quality (Table 3). Yield of marketable No.1
fruit in plots treated with chlorothalonil after the accumulation of every 20 or 25
DSVs was similar to plots sprayed weekly (Table 4B). Percent No.1 fruit was similar
in the weekly, DSV 20L, and DSV 25H treatments. Yield and percent of No.1 fruit
was compromised in plots not sprayed as compared to plots sprayed weekly. With
regard to weight of cull fruit, the interaction of frmgicide x tillage was signiﬁcant
(Table 3). Weight of culls decreased in the no spray, DSV 20L, DSV 20H, DSV 25L
and DSV 25H treatments, and increased in DSV 20H and weekly treatments in ZT as
compared to CT plots (Table 4C). CT plots sprayed weekly had the lowest weight of

culls. Total yield was not affected by fungicide treatment (Figure 2).

 

 

TABLE 3: Mean 5

 

Source
of
Variability

Rep.

Tillage (T)

Error a

 

Fume. (F)

K

'l‘xF

 

Error b

§

 

1,11,“! F-test Signii
NS, non-sir
xlirllit were sorta
y 1990 Was the f.
(R. different t1

TABLE 4A. 1

 

 

 

128

TABLE 3: Mean squares from analysis of variance for yield and fruit quality of fresh
market tomato (FMT) in 1990.

 

 

 

 

 

 

 

 

 

 

 

 

 

Souirce FMT FRUIT QUALITY AND YIELD (MT/HA)"
O

variability df LARGE #1 %LRG #1 MED #1 # 2 CULL
Rep. 7y 136 NS 367 ** 6O * 464 ** 123 NS
Tillage (T) 1 528 NS 138 NS 60 NS 472 * 17 NS
Error a 7 124 62 14 50 77
Fung. (F) 6 143 * 102 ** 18 NS 45 NS 68 *
T x F 6 35 NS 48 NS 6 NS 20 NS 87 **
Error b 84 58 27 11 41 26

 

 

 

 

 

 

iii *“

, , F—test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
NS, non-signiﬁcant.

" Fruit were sorted for large #1, % large #1, medium #1, #2 and culls.

y 1990 was the ﬁrst year of the experiment resulting in 8 replications per treatment

(i.e. different treatments were not applied to the main rotation plots).

TABLE 4A. Effect of tillage on marketable yield (metric tonnes) of fresh market
tomato in 1990.

 

 

 

 

 

 

 

 

 

LARGE NO.1 MT/Ha
TILLAGE
MT/Ha % MEDIUM No.2 CULL
NO.1
L CT 33.6 39 9.9 23.9 19.3
L ZT 29.3 37 11.4 19.8 186
D VALUE 0.08 0.18 0.08 0.02 0.6

 

 

 

 

 

 

TABLE 43. Effect

 

FUNGICID?
TREATMET
WEEKLY
nsv zap
osv 20H
nsv 25L
nsv 25H
N0 SPRA‘.

P VALUE

‘ means followed 1
011 LSD (P=0.05,

 

 

’ interaction signjf

Dream fOllowed
on LSD (

 

129

TABLE 48. Effect of fungicide treatment on marketable yield (metric tonnes) of fresh
market tomato in 1990.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

FUNGICIDE LARGE NO.1 MT/Ha
TREATMENT MT/Ha % MEDIUM No.2 CULL
WEEKLY 34.5 AK 42 A 10.8 21.2 15.3
DSV 20L 34.5 A 39 AB 11.3 23.5 21.2
DSV 20H 30.9 AB 38 B 11.2 21.4 18.3
DSV 25L 32.5 A 37 BC 10.4 24.0 21.4
DSV 25H 28.8 AB 39 AB 8.4 19.0 18.9
NO SPRAY 26.3 B 34 C 10.9 21.5 19.3
P VALUE 0.03 0.002 0.14 0.37 i
‘ means followed by the same letter within a column are not significantly different based

on LSD (P=0.05, n=8).

y interaction signiﬁcant (P=0.01).

TABLE 4C. Means of cull weight of fungicide x
tillage interaction of fresh market tomato in 1990.

FUN GICIDE
TREATMENT

 

" means followed by the same letter across both columns are not signiﬁcantly different
based on LSD (P=0.05, n=8).

 

 

FIGURE 1:]
harvested eat

 

 

130

FIGURE 1: Effect of tillage and rotation on fresh market cv. ’Pik-Rite' tomato yields
harvested each week.

 

 

KG PER PLOT

KG PER PLOT

KG PER PLOT

131

 

 

30-

 

:3:— CONV. TILLAGE

ZONE TILLAGE

     

1990

 

 

 

 

 

O I J l l
AUG 7 AUG 15 AUG 22 AUG 28 SEP 5 SEP 12
30

 

20-

10-

 

 

% CONV. TILLAGE]

ZONE TILLAGE

1991

 

 

 

 

 

 

 

  
  
  

 

 

O I I I I 1
JUL 30 AUG 5 AUG 12 AUG 21 AUG 27
40- —E— NO ROTATION & CT 1 992 —
_ {E NO ROTATION & ZT
-§— ROTATION & CT _
30-
3: ROTATION & ZT

 

AUG 19

 

 

    

 

 

I
AUG 25 SEP 01

 

 

I l 1
SEP 09 SEP 15 SEP22

DATE OF HARVEST

 

 

FIGURE 2
week

 

 

132

 

FIGURE 2: Effect Of fungicide on fresh market cv. ’Pik-Rite’ tomato yields harvested each
week.

 

 

 

KG PER PLOT

KG PER PLOT

KG PER PLOT

 

 

133

 

 

   
  
 
   
  
    
   

 

 

404 {E— WEEKLY -
1 DEV 20L
:51 osv 20H

30" '-
Z st 25L
‘ % osv 25H

202 Lil NO SPRAY -

 

 

 

 

 

 

 

 

 

 

  
   
  
  
 
    
 
  
 
  
  
  
   

 

 

 

AUG7 AUG15 AUG22 AUG28 sei=5 SEP12
30
WEEKLY
4 if: 1991 L
£— DSV15L
20 . f DSV15H
1 -} st 20L _
-3_E- stzoI-I
& NO SPRAY
10- L

 

 

 

 

 

 

 

 

 

o l l I l 1
JUL 30 AUG 5 AUG 12 AUG 21 AUG 27
40- f WEEKLY r
4 —_,I_- osv 15 L
30 {- DSV15H
1 _
—£— osv 20L
‘ %— st 20H
204 -} DSV 25 H -
i NO SPRAY L
10- -

 

     

 

 

—"'"'_' r I I
AUG 19 AUG 25 $61; 01 SEP 09 SEP 15 SEP22
DATE OF HARVEST

l
weudur
observa
ofchhu

Sprayed

1991 S
PRT h.
to plat
and C

16W ﬂt

PRT:
plant
plots
on Ft
The I
grow

1991

plots

20H

 

 

134

In the case Of processing tomatoes (cv. Ohio 7870) tillage impacted the total
weight of green fruit and % ripe fruit harvested (Table 5 and 6). Based on the latter
observations, ZT appeared tO delay fruit maturity during the 1990 season. Applications
Of chlorothalonil did not impact fruit yield nor fruit quality as compared to plots not

sprayed (Table 5 ).

1991 SUMMER SEASON - Assessment of plant phenology and growth: Mean
PRT height on Jun 19, 1991 was greater (P=0.05) in ZT plots (28.6 cm) as compared
tO plants in CT plots (26.5 cm). FMT plants were 39.8 and 36.2 cm (P=0.017) in ZT
and CT plots, respectively. Number Of FMT ﬂowers was not affected by tillage. TOO
few ﬂowers were set on PRT plants by Jun 19.

Tillage did not affect plant height or number of fruit set per plant of FMT and
PRT as recorded on Jul 3 1991 (data not shown). Mean number Of ﬂower clusters per
plant did not differ on FMT plants but were lower (P=0.007) on PRT plants in ZT
plots (number = 11.0) as compared to CT plots (number = 13.4). Mean fruit diameter
on FMT plots was 3.7 cm and 4.8 cm (P=0.02) in ZT and CT plots, respectively.
The number Of chlorothalonil sprays applied in 1990 appeared to impact tomato
growth in 1991. Prior tO any applications in 1991, mean PRT plant height Jun 19,
1991 in plots sprayed with 15 weekly applications in 1990 was 26.4 cm, signiﬁcantly
less than plant height Of 29.7 cm in plots not sprayed during 1990. Plant height in
plots sprayed 4 times after the accumulation Of every 20 disease severity values (DSV
20H treatment) in 1990 was intermediate at 27.8 cm (LSD = 2.5 cm, 12 df, n=8). By

Jul 3, differences were more pronounced. Mean plant height in the weekly (now with

 

 

 

 

TA

 

TA]

 

135

TABLE 5: Mean squares from analysis of variance for yield and fruit quality of
processing tomato (PRT) in 1990.

 

 

 

 

 

 

 

 

 

Source PRT FRUIT QUALITY AND YIELD
of (MT/HAY
Variability df
WT RIPE WT WT % RIPE
GREEN CULL
Rep. 7y 1290 NS 64 NS 82 NS 213 NS
Tillage (T) 1 608 NS 636 ** 4 NS 572 *
Error a 7 1228 42 58 71
Fungicide (F) 6 94 NS 66 NS 64 NS 108 NS
T x F 6 372 NS 60 NS 62 NS 101 NS
Error b 84 247 44 29 61

 

 

 

 

 

 

 

 

* ii iﬁt

, , F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
NS, non-signiﬁcant.

" Fruit were sorted for color (ripe or green) and quality (marketable fruit or culls).

’ 1990 was the ﬁrst year Of the experiment resulting in 8 replications per treatment
(i.e. different treatments were not applied to the main rotation plots).

TABLE 6: Effect Of tillage on marketable yield of processing tomato cv. OHIO 7870

 

 

 

 

 

 

 

 

 

in 1990.
% RED
CULLS
7.0 86
7.4 82
<0.30 0.03

 

 

 

 

 

 

 

3 applicatir
was 50.9, :
between ti

0n
sampled fr
weight, to
weight am

plants bar

(104 gms]

Tomato 1
plots but
plots (Fig
grade sizI
(Table 81
effects (1]

Ir
(Table 91
Variance

 

 

 

136
3 applications in 1991), DSV 20H (no applications in 1991) and no spray treatments

was 50.9, 56.2, and 56.8 cm, respectively (LSD=4.7 cm, 12 df, n=8). The interaction

 

between tillage and fungicide treatment was not signiﬁcant.

On Aug 9 1991, one PRT plant outside the ﬂagged region was destructively
samﬂed from each plot of the weekly, DSV 20H and no spray treatments. Fresh
weight, total number of leaves, number of fruit, total and mean weight of fruit, and
weight and percent of ripe fruit was not affected by tillage. Whole plant dry weight Of
plants harvested from ZT plots was less (78 gms) as compared to plants from CT plots

(104 gms) (P=0.05). NO difference due to fungicide treatment was Observed.

Tomato fruit yield and quality 1991: Early yield was not impacted by ZT in FMT
plots but by the third harvest weekly yields were less in ZT plots compared to CT
plots (Figure 1). Yield decline in ZT plots was not signiﬁcant at P = 0.05 for each
grade size (Table 7). Reduced yield Of No.1 fruit was marginally signiﬁcant at P=0.06
(Table 8A). Plots sprayed weekly tended to have the lowest yields but no signiﬁcant
effects due to fungicide treatment were Observed (Table 7, 8B).

In the case of processing tomato (cv. Heinz 8704) tillage did not affect yields

 

(Table 9, 10). Fungicide did not affect total yields (data not shown) but analysis of
variance revealed weight of green fruit was affected. One Of the two control plots had

more green fruit than any other treatment (data not shown).

 

 

TAB

137

TABLE 7: Mean squares from analysis Of variance for yield and fruit quality of fresh
market tomato (FMT) in 1991.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Soupce FMT FRUIT QUALITY AND YIELD (MT/HA)x
O

variability df LARGE %LRG MED # 2 CULL

#1 #1 #1

Rep. 3y 84 NS 52 NS 65 NS 126 NS 25 NS
Tillage (T) 1 719 NS 227 NS 82 NS 558 NS 5 NS
Error a 3 79 89 19 119 21
Fung. (F) 6 63 NS 55 NS 16 NS 50 NS 13 NS
T x F 6 37 NS 30 NS 9 NS 38 NS 20 NS
Error b 36 40 39 12 46 15

 

***

NS, non-signiﬁcant.

" Fruit were sorted for size and quality

, f“ F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.

V 1991 was the second year of the experiment with half the plots planted to tomato
(4 reps) and half to cucumber.

 

 

 

 

TA

 

TAE

 

 

TABLE 8A. Effect of tillage on marketable yield Of fresh market tomato in 1991.

138

 

 

 

 

 

 

 

 

 

 

 

LARGE NO.1 MT/Ha
TILLAGE
MT/Ha % MEDIUM NO.1 NO.2 CULL
CT 22.1 31.6 13.2 18.9 15.0
ZT 15.0 27.6 10.8 12.6 14.4
P VALUE 0.06 0.21 0. 13 0.1 1 <0.30

 

 

TABLE SB. Effect of fungicide treatment on marketable yield of fresh market tomato

 

 

 

 

 

 

 

 

 

in 1991.
FUNGICIDE LARGE NO.1 MT/Ha
TREATMENT
MT/Ha % MEDIUM NO.1 NO.2 CULL
WEEKLY 16.1 29 10.3 18.4 13.2
DSV 20L 21.3 31 12.5 14.2 14.4
DSV 20H 19.8 29 - 12.0 18.7 15.3
DSV 25L 21.3 33 11.1 17.6 14.7
DSV 25H 20.5 32 15.0 15.0 13.0
NO SPRAY 16.6 28 11.8 13.4 16.1
P VALUE 0.19 0.23 0.19 <0.30 0.26

 

 

 

 

 

 

 

 

 

 

 

 

TA]

 

139

TABLE 9: Mean squares from analysis of variance for yield and fruit quality of
processing tomato (PRT) in 1991.

 

 

 

 

 

 

 

 

 

Source PRT FRUIT QUALITY AND YIELD (MT/HA)x
Vaﬁzbility df WT RIPE WT WT % RIPE
GREEN CULL

Rep. 3y 273 NS 251 NS 84 NS 423 NS
Tillage (T) 1 1154 NS 323 NS 42 NS 1 NS
Error a 3 861 172 23 209
Fungicide (F) 6 290 NS 122 ** 19 NS 44 NS
T x F 6 61 NS 52 NS 23 * 71 NS
Error b 36 141 29 8 68

 

 

 

 

 

 

 

at ** iii

, , F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
NS, non-s1 gniﬁcant.

" Fruit were sorted for color (ripe or green) and quality (marketable fruit or culls).

3’ 1991 was the second year Of the experiment with half the plots planted to tomato
(4 replications) and half to cucumber.

TABLE 10: Effect Of tillage on marketable yield Of processing tomato cv. Heinz 8780

 

 

 

 

 

 

 

in 1991.
MT/Ha
TILLAGE % RED
RIPE GREEN CULLS
C1“ 46.9 21.1 6.5 63
ZT 37.8 16.3 4.8 63
P VALUE <0.30 0.26 0.27 <0.3O

 

 

 

 

 

 

 

 

Cucu:
exact
Howe
likely
treatr
Then
comp
were
estin

MlS'

 

140

Cucumber fruit yield 1991: The goal of the ZT system was to seed cucumber on the
exact same row center where tomato once stood without further tillage inputs.
However, soil pent-rometer readings in the early spring indicated a repeated ZT would
likely beneﬁt the cucumber crop. Therefore, zones were again tilled. Fungicide
treatments to tomato during 1990 did not impact yield of cucumber during 1991.
There-fore, data from sub—sub-plots were pooled and analyzed as a randomized
complete block design with 1 factor (tillage). Cucumber yield in ZT and CT plots
were similar (Table 11). Four cucumber plots were affected by a broken tile and
estimated values for each grade size was determined using the MSTAT-C

MISVALEST and used for analysis Of variance and reporting of means.

TABLE 11: Effect Of tillage on yield Of pickling cucumber cv. Flurry in 1991.

 

 

 

 

 

GRADE SIZE (MT/Ha)"
TILLAGE
NO.1“ No.2x NO.3y No.42 CULLS TOTAL
CT 0.30 1.0 3.8 3.5 0.8 i 9.4
ZT 0.33 1.1 4.5 3.3 0.9 10.1
P VALUE NS NS NS NS NS NS

 

 

 

 

 

 

 

 

 

" means over CT plots include 4 estimated values for plots that were over a broken
ﬁeld tile. Based on the original data, mean weight Of NO. 3 fruit in ZT plots was
signiﬁcantly greater as compared to CT plots. Substituting estimated values did not
affect signiﬁcant differences among any other set Of means including total weight.

“' Pickling cucumbers <2.7 cm in diameter
" Pickling cucumbers between 2.7 and 3.8 cm in diameter
3' Pickling cucumbers between 3.8 and 5.1 cm in diameter
‘ Pickling cucumbers >5.1 cm in diameter.

 

1992 SUV
mean plan
with cucu
plant not
mean plat
treatment:
each plot
signiﬁcar

weight 0:

Tomato
yield on
(i.e. 1 dl
for each

are high

2) but u
marketa
Table 1
Selects:

II10del I

 

141

1992 SUMMER SEASON - Assessment of plant phenology and growth: FMT
mean plant height was greater (49.7 cm vs 46.4 cm; P=0.02) on Jun 18 in plots rotated
with cucumber as compared to plots not rotated. No differences in other estimates of
plant productivity were observed for FMT plants nor PRT plants on Jun 18. On Jul 17,
mean plant height and number of ﬂower clusters was highest in ZT x rotation
treatments of PRT plants. One PRT plant was destructively sampled on Aug 13 from
each plot of 3 sub—sub-treatments similar to the method described for 1991. No
signiﬁcant difference were observed in number or weight of fruit set, in total fresh

weight or dry weight.

Tomato fruit yield and quality 1992: Rotation combined with zone tillage increased
yield on speciﬁc harvest dates of FMT tomato (Figure 1). With only 2 replications
(i.e. 1 df for the numerator and denominator) signiﬁcant differences were not apparent
for each fruit quality category (Table 12). The yields as affected by rotation and tillage
are highlighted in Table 13A.

Application of fungicide impacted FMT yield on speciﬁc harvest dates (Figure
2) but no differences were Observed for total yield harvested (data not shown) nor for
marketable fruit. The weekly, DSV 25H and no spray treatment means are listed in
Table 13B. The interaction of fungicide x rotation treatment was signiﬁcant and
selected results are outlined in Table 13C. The ANOVA and yield data for the full

model with 4 replications is provided in Appendix A.

 

TAB?

 

 

 

 

TABLE 12: Mean squares from analysis of variance for yield and fruit quality of fresh
market tomato (FMT) in 1992.

142

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Source FMT FRUIT QUALITY AND YIELD (MT/HA)x

Vaﬁgiﬂity df LARGE #1 %LRG #1 MED #1 #2 CULL
Rep. 1y 69 NS 94 NS 3.3 NS 1645 NS 0.1 NS
Rotation (R) 1 682 NS 232 NS 3.7 NS 213 NS 52 NS
Error 3 1 130 203 0.3 452 18
Tillage (T) l 10 NS 28 NS 6.7 NS 288 NS 10 NS
R x T 1 17 NS 102 NS 9.8 NS 206 NS 281 *
Error b 2 73 126 3.5 199 8
Fungicide (F) 6 52 NS 33 NS 4.6 NS 113 NS 20 *
R x F 6 25 NS 32 NS 7.2 NS 47 NS 30 i
T x F 6 60 NS 40 NS 5.2 NS 29 NS 5 NS
R x T x F 6 57 NS 23 NS 4.8 NS 54 NS 12 NS
Error c 24 38 37 2.3 66 8

 

 

 

 

 

 

 

 

 

* it ***

, , F-test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
NS, non-signiﬁcant.

‘ Fruit were sorted for large #1, % large #1, medium #1, #2 and culls.

3’ nearly all sub-sub—plot treatments in 2 replications of a sub-plot treatment (conventional
tillage) of a main-plot treatment (tomato rotated to cucumber) were adversl y affected
by a ﬁeld tile that malfunctioned, resulting in poor drainage. Therefore the data were
analyzed over 2 replications instead of 4 (n=56, not 112).

 

 

 

TAB:

 

TAE

 

 

143

TABLE 13A: Effect Of rotation and tillage on marketable fruit of fresh market tomato

 

 

 

 

 

 

 

 

 

in 1992.
LARGE NO.1 MnHa
MT/Ha % MED #1 No.2 CULL
NO ROTATION 18.6 2; 7.9 21L 17.0
WITH ROTATION 25.6 31 8.4 31.1 18.9
P VALUE 0.;6_ <0.30 ' 0.16 <0.30 <0.30
CT 12117 29 7.8 26.9 17.5
ZT 12.5 28 8.5 31.4 18.4
P VALUE <0.30 <0.30 0.30 <0.30 <0.30

 

 

 

 

 

 

 

 

TABLE 13B. Effect Of fungicide treatment on marketable yield of fresh market tomato

 

 

 

 

 

 

 

 

 

 

 

 

in 1992.
FUNGICIDE LARGE No.1 MT/Ha
MT/Ha % MED#1 No.2__ CULL
WEEKLY 2_2.6 30.9 6.9 29.0 14.9
st 25H 25.2 30.8 8.0 30.5 18.4
NO SPRAY 19.3 26.5 8.3 g3_.5 19.2
P VALUE 0.26 <0.30 0.10 0.16 Y

 

 

 

 

Y Rotation x Fungicide interaction was signiﬁcant.

TABLE 13C: Means of cull fruit of selected fungicide x rotation treatment

 

 

 

 

 

 

 

 

 

interactions.
CULLS MTlHa
NO ROTATION WITH ROTATION -
WEEKLY 16.8 13.0
DSVﬁI 13.9 r_2_2.9
NO SPRAY 18.9 19.5
LSD (n=4) ﬂ 4.0

 

 

 

 

 

weather
prematI
grand 1
of mar.
47.2 N.
combir
and 37
was 1e
with c

l), but

and p
same
rotati
199C
like]
less,
(Fig
OPP

mus

 

 

 

144

Ethrel was applied on PRT plants Sep 14 to enhance ripening. However, cool
weather persisted and Ethrel appeared to have little impact. The experiment was
prematurely terminated on Sep 29 to avoid forecasted frost and loss of yield data. The
grand mean of percent ripe fruit was 24.5%. Therefore, total yield is presented instead
of marketable ripe fruit. Total yield in rotation plots was 43.3 MT Ha'l as compared to
47.2 MT Ha‘l in plots planted to continuous tomato (P=0.27). ZT increased yield in
combination with all other treatments. The mean yield was 53.0 MT Ha'l in ZT plots
and 37.4 MT Ha‘l in CT plots (P=0.04). All interactions were not signiﬁcant. Yield
was less in plots not sprayed (35.1 MT Ha") as compared to plots sprayed weekly
with chlorothalonil (mean = 48.9 MT Ha") and the DSV 25H treatment (48.2 MT Ha’

1), but the F-ratio for fungicide effect was marginally signiﬁcant at P=0.06.

DISCUSSION

The zone tillage system enhanced or did not affect total yield of fresh market
and processing tomato in 1990, did not affect yield of cucumber planted on the exact
same row center in 1991, and enhanced yield of tomato in 1992 when combined with
rotation. Our goal was to perform no additional tillage operations from the spring of
1990 to the summer Of 1991. However, soil density readings indicated beneﬁt would
likely occur with a repeat tillage Of the zone prior to planting cucumber. Never-the-
less, compared to a conventional tillage system, 4 CT operations were eliminated
(Figure 1 of Chapter 2). Immediately after the cucumber harvest, a window of
Opportunity for a summer fallow (Sarrantonio 1992) was exploited to seed a green

mustard crop. The crop was chosen for its possible ability to reduce inoculum of plant

 

 

pathoger
protect t
nutrient:
included
determir
convent
weed, i
the putt

no-till :

the ent
apply I
reduce
Hoyt 1

rye Wt

 

 

145

pathogens (Mojtahedi et a1. 1993; Muehlchen et al. 1990), for its rapid growth to
protect the light sandy soils from erosion, and for its potential ability to tie up
nutrients that other wise may leach from the soil (Sarrantonio 1992). The crop was
included as part of a production system and the experiment was not designed to
determine potential beneﬁts of the mustard crop. After the mustard crop, complete
conventional tillage was performed. In this manner, as opposed to continuous no-till,
weed, insect and pathogen life cycles can be disrupted on a biennial cycle to reduce
the potential of serious build up of pests in the vegetable production system. Complete
no-till systems may be unrealistic for vegetable crops.

The zone tillage system also provided a surface rye residue that persisted for
the entire cropping season. Most advantages associated with no-till would therefore
apply to this zone tillage system including reduced water and wind soil erosion,
reduced wind-wiping of plants, and enhanced water use efﬁciency, etc (Coolman and
Hoyt 1993; Gebhardt et al. 1985; Phillips et a1. 1980). Likewise, the known beneﬁts of
rye were exploited to smother weed and, through allelopathic substances, delay early
season weed emergence and the need for early season herbicide (Putnam 1990;
Wallace and Bellinder 1992). Most important, the preparation of strips early in the
season, combined with zone tillage, circumvented problems commonly associated with
nO-till systems in vegetable production (Doss et al. 1981; Knavel et a1. 1977; Price and
Baughan 1988). For example, Price and Baughan ( 1988) have shown tomato
transplants set into nO-till plots are less productive than plants in conventional tillage
(CT) plots. Reduced productivity may be due to allelopathic substances released by rye
residue, high carbon to nitrogen ratio limiting early season nitrogen availability, poor

root to soil contact of the plant, or possibly high populations of parasitic pathogens

 

 

 

that pets
not acct
time for

avoided

problen
product
market
on Jun
PalaIIIt
On At
compa
positit
yields
weeld
produ
inside
know

for p

TON
at re
0011):

lick

 

 

 

146

that persist in association with rye residue. This ZT system ensures rye residue does
not accumulate in strips where plants are to go. Early season kill of strips also allows
time for decomposition of the residue that overwinters and potential disadvantages are
avoided.

ZT fractures the soil where plants are to go and ensures compaction is not a
problem. ZT loosens the soil for enhanced root exploration and subsequent plant
productivity (Grajauskis 1990). In 1990, ZT allowed for total yields of both fresh
market and processing tomato similar to a CT system. In 1991, initial measurements
on Jun 19 indicated enhanced plant productivity in ZT plots. However, by Jul 3,
parameters of plant productivity began to indicate ZT plants lagged behind CT plants.
On Aug 9, total dry weight of plants harvested from ZT plots was signiﬁcantly less as
compared to CI‘ effects. Ultimately, yields were substantially reduced (Figure 1). The
positive effect of ZT in 1991 on early plant productivity is supported by similar initial
yields in ZT and CT plots for the ﬁrst two FMT harvest dates, after which time
weekly yields in CT plots surpassed yields in ZT plots (Figure l). The decline in plant
productivity can be associated with the lack Of rotation. Plants in ZT plots had a high
incidence of collar rot (Chapter 2). Lesions completely girdled stems, a problem
known to decrease plant productivity (Jones et al. 1991). Also, lack of tillage allowed
for persistence of weeds which became problematic during the growing season.

Applications of chlorothalonil on a reduced schedule, according to the
TOMCAST model, can reduce control of defoliation due to early blight and, especially
at reduced rates of fungicide, can increase the incidence Of fruit mold (Chapter 2) as
compared to plots sprayed weekly. However, reduced sprays rarely affected marketable

yield of processing and fresh market tomato. One notable exception occurred with

 

 

FMT in 1
substantit
been asst
(Chapter
sprays pt

I
sprays h
Brarmna
accordir
our stud
occasior
not to s
superior

future,

yield (1

tended

Satire;

PIOduc

cides,

Piodm

 

 

147

FMT in 1990. CT combined with weekly sprays limited weight of culls most
substantially compared to other fungicide treatments. The high incidence of culls had
been associated with a high incidence Of soil rot, caused by Rhizoctonia solani
(Chapter 2). Soil rot problems did not persist and were controlled with a reduced-
sprays program in subsequent years (Chapter 2).

Lack of association of yield decline with defoliation and amount of fungicide
sprays has been noted by others (Brammall 1993; Ferrandino and Elmer 1992).
Brammall demonstrated marketable yield was not impacted by chlorothalonil, applied
according to the TOMCAST model, as compared to plots not sprayed. However, in
our study, plots not sprayed tended to have the lowest marketable yield and on
occasion, such as PRT yields in 1992, were close to signiﬁcance. Recommendations
not to spray would introduce considerable risk at this time. However, if lines with
superior genetic resistance can be incorporated into the production system in the
future, cultural and genetic control may suffice.

Fungicide applications on a weekly schedule never resulted in highest total
yield (Figure 2). In fact, plants in plots sprayed weeldy were stunted in 1991 and yield
tended to decrease. Fifteen fungicide sprays were applied the preceding year to the
same plots. However, evidence proving a direct link was not acquired.

The ZT system outlined here has considerable potential for vegetable
production systems. Additional research is required to determine optimum rotation
cycles, the potential of using ZT with other crops, and to build up a larger knowledge
base that will enable growers to ﬂexibly manage on farm inputs for enhanced crop

productivity and proﬁtability.

 

 

 

 

148

LITERATURE CITED

Abdul-Bald, A.A., and JR. Teasdale. 1994. A nO-tillage tomato production system
utilizing hairy vetch and subterranean clover mulches. HortScience: in press.

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

Coolman, R.M., and GD. Hoyt. 1993. The effects of reduced tillage on the soil
environment. HortTechnology 3: 143-145.

Doss, B.D., J.L. Turner, and CE. Evans. 1981. Inﬂuence of tillage, nitrogen, and rye
cover crop on growth and yield of tomatoes. J. Amer. Soc. Hort. Sci. 106:95-97.

Ferrandino, F.J., and W.H. Elmer. 1992. Reduction in tomato yield due to Septoria
leaf spot. Plant Dis. 76:208-211.

Frye, W.W., and R.L. Blevins. 1989. Economically sustainable crop production with
legume cover crops and conservation tillage. J. Soil Water Conserv. 44:57-60.

Gebhardt, M.R., T.C. Daniel, E.E. Schweizer and RR. Allmaras. 1985. Conservation
tillage. Science 230:625-630.

Grajauskis, 1.]. 1990. Effects of nitrogen, rye cover and zone tillage on the yield of
fresh market tomatoes in three tillage systems. MS Thesis. Michigan State University.

70 pp.

Jones, J.B., J.P. Jones, R.E. Stall, and T.A. Zitter. 1991. Compendium of Tomato
Diseases. APS Press, St. Paul, MN.73pp.

Kelly, W.C. 1990. Minimum use of synthetic fertilizers in vegetable production.
HortScience 25:168-169.

Knavel, D.E., J. Ellis, and J. Morrison 1977. The effects of tillage systems on the
performance and elemental absorption by selected vegetable crops. J. Amer. Soc. Hort.
Sci. 102:323-327.

Little, T.M., and F.J. Hills. 1978. Agricultural Experimentation: Design and Analysis.
John Wiley and Sons, New York, p.350.

McKeown, A.W., R.F. Cerkauskas, and J.W. Potter. 1988. Inﬂuence of strip tillage on
yield, diseases and nematodes of tomatoes. J. Amer. Soc. Hort. Sci. 113:328-331.

Mojtahedi, H., G.S. Santo, J.H. Wilson, A.N. Hang. 1993. Managing Meloidogyne
chitwoodi on potato with rapeseed as green manure. Plant Dis. 77:42-46.

 

 

 

 

149

Muehlchen, A.M., R.E. Rand, and J.L. Parke. 1990. Evaluation of crucifer green
manures for controlling Aphanomyces root rot of peas. Plant Dis. 74:651-654.

Phillips, R.E., R.L. Blevins, G.W. Thomas, W.W. Frye, and SH. Phillips. 1980. No-
till agriculture. Science 208:1108-1 113.

Price, H.C., and RA. Baughan. 1987. Establishment of fresh market tomatoes in a no-
till system. Acta Hort. 198:261-268.

Putnam, AR. 1990. Vegetable weed control with minimum herbicide inputs.
HortSicence 25:155-159.

Sarrantonio, M. 1992. Opportunities and challenges for the inclusion of soil-improving
crops in vegetable production systems. HortScience 27:754-758.

Shelby, P.P.,Jr., D. L. Coffey, N. Rhodes, Jr., and LS. Jeffery. 1988. Tomato
production and weed control in no-tillage versus conventional tillage. J. Amer. Soc.
Hort. Sci. 113:675-678.

Sloneker, LL, and W.C. Moldenhauer. 1977. Measuring the amounts of crop residue
remaining after tillage. J. Soil and Water Conserv. 32:231-236.

Wallace, R.W., and RR. Bellinder. 1992. Alternative tillage and herbicide options for
successful weed control in vegetables. HortScience 27:745-749.

 

 

 

 

CHAPTER IV

MAJOR DISTINCTIONS IN GENONIIC STRUCTURE DETECTED BY REP-
PCR FINGERPRINTING SEPARATE STRAINS CLASSIFIED AS
XANTHOMONAS CAMPESTRIS PV. VESICATORIA INTO AT LEAST FOUR

GROUPS.

 

 

150

   

 

elemen
that id:
pv. ves
migrat:
origin;
starch
relativ
isolate
pectol
the t0
Amer
as Gn
item
with

camp

 

 

 

 

151

ABSTRACT

DNA primers corresponding to repetitive sequences (REP, BOX and ERIC
elements) and the PCR (rep-PCR) were used to generate complex ﬁngerprint patterns
that identiﬁed 4 distinct groups among strains classiﬁed as Xanthomonas campestris
pv. vesicatoria. These groups were differentiated by near complete dissimilarity in
migration rates of 60 or more bands generated with rep-PCR. Group A isolates
originated from tomato or pepper. Most of these isolates proved to be negative in
starch hydrolysis and pectolytic activity tests. All Group A isolates were found to be
relatively homogenous with regard to their rep-PCR ﬁngerprinting patterns. Group B
isolates originated primarily from tomato and were positive for starch hydrolysis and
pectolytic activity. Group B strains were found to comprise an important component of
the tomato spot complex in the northcentral tomato production region of North
America. One isolate was classiﬁed as a Group C strain. Two isolates were classiﬁed
as Group D strains and one such isolate was found to be highly virulent to tomato.
Interestingly, group D strains were found to share numerous bands of similar mobility
with strains pathogenic for cabbage, classiﬁed as Xanthomonas campestris pv.

campestris, suggesting the group D strains are closely related to the cabbage pathogen.

 

 

 

spot on

Ganhm
andt01
tonuto
losses
Chenﬁ
nmnag
smart
andIK
badsr

ofcrc

QMHI
dhea
ﬂute
deve

thei

due

 

 

 

152
INTRODUCTION

Xanthomonas campestris pv. vesicatoria (Xcv), the causal agent of bacterial
spot on pepper and tomato, was ﬁrst diagnosed in the early 1920’s (Doidge 1921;
Gardner and Kendrick 1921; Higgins 1922) and occurs worldwide in regions of pepper
and tomato production (Hayward and Waterson 1964; Sherf and MacNab 1986). On
tomato, Xcv affects all above ground plant tissue and can incite marketable yield
losses from 5 to 70% (Pohronezny and Volin 1983; Sherf and MacNab 1986).
Chemically based and cultural practices are currently the primary farm—level disease
management strategies. However, routine application of bactericides, such as copper or
streptomycin, do not provide consistent control, because of low efﬁcacy (Hausbeck
and Kusnier III 1993) and the ability of populations to acquire resistance to the
bactericides (Minsavage et al. 1990; Stall et al. 1986). Cultural practices such as burial
of crop debris, crop rotation and use of windbreaks to limit on—farm incidence of spot
in tomato has been recommended (Jones et a1. 1991; Sherf and MacNab 1986) and
implemented, but nevertheless throughout the northcentral region of North America
(MI, OH, IN, USA. and Ontario, Canada), spot problems recur each year. Farm level
integrated disease management practices appear to have minimal impact on disease
control, especially when weather conditions favor the spread of disease. Ultimately,
disease control is likely to be achieved primarily through disease management
strategies implemented before the seed (or transplants) anive at the farm, such as the
development of genetic resistance and implementation of protocols designed to limit
the introduction of the initial inoculum (Goode and Sasser 1980).

However, breeding for durable disease resistance and implementing necessary

detection/diagnostic protocols have posed a challenge because Xcv is phenotypically,

 

 

 

153
serologically, pathogenically and genotypically diverse (Doolittle and Crossan 1959;
Dye 1962; Dye et al. 1964; Jones et al. 1993a; Minsavage et al 1990.; Klement 1959;
Stall et al. 1993; Sutic 1959; Vauterin et al. 1990; 1991; Wang et al. 1990; Whalen et
al. 1988). For example, genetic resistance was developed in tomato (Scott et al. 1989),
but a strain from Argentina and virulent for the resistant host was already identiﬁed
(Wang et al. 1990) before the resistance was commercially deployed.

Techniques that emphasize overall chromosomal organization of Xcv may help
elucidate our understanding of the genotypic structure of natural populations and may
provide a framework for understanding the evolutionary dynamics of pathogenesis and
optimal mathods for the implementation of integrated disease management strategies,
including diagnostic protocols, plant breeding programs, and the deployment of genetic
resources. Therefore, this research was initiated using a genomic DNA ﬁngerprinting
approach to determine the genetic diversity of Xcv isolates obtained from diverse
geographic regions. This rapid and highly reproducible method employs primers
corresponding to repetitive extragenic sequences [repetitive extragenic palindromic
(REP) sequences (Gilson et al. 1984; Higgins et a1. 1982), enterobacterial repetitive
intergenic consensus (ERIC) sequences (Hulton et al. 1991; Sharples and Lloyd 1990),
and the BOX element (BOX 1A sequences) (Martin et al. 1992)] to generate complex
ﬁngerprint patterns from DNA of bacteria in combination with the polymerase chain
reaction (PCR) protocol (V ersalovic et al. 1991; de Bruijn 1992; Koeuth et al. 1993).
The technique, known as REP-PCR, ERIC-PCR and BOX-PCR, respectively (and rep-
PCR collectively), distinquishes Xanthomonas and Pseudomonas strains at the pathovar

and subpathovar level (see Appendix D), presumably based on overall chromosomal

organization.

 

 

 

 

 

 

 

154

This chapter highlights the detection of 4 distinct groups among isolates
classiﬁed as Xanthomonas campestris pv vesicatoria. The groups correlate with
selected phenotypic characteristics such as amylolytic and pectolytic activity,
highlighting the utility of rep-PCR in distinguishing phytopathogenic bacteria at the
pathovar and sub-pathovar level. This chapter also shows that amylolytic/pectolytic
strains [subgroup ”B” strains sensu Vauterin et al. (1990) and Group ”B” or T2 strains
sensu Jones et al. (1993)] constitute an important component of the tomato spot

complex in the northcentral production region of North America.

MATERIALS AND METHODS

BACTERIAL ISOLATES AND CULTURE CONDITIONS. MSU accession
numbers, original strain designation(s), geographic origin, year of isolation, race
designations and sources of bacterial isolates or genomic DNAs are listed in Table 1.
Xcv suspensions initiated from single colonies were stored at -70 C in 15% glycerol
and re-streaked on nutrient-yeast—dextrose agar (NYDA) (Jones et a1. 1981) as
required. Bacterial cells were grown for DNA isolation from single colonies in 40 m1

LB for 24 to 48 hr at 27°C on a rotary shaker (200 rpm).

ISOLATION OF CHROMOSOMAL DNA AND PCR CONDITIONS. Total

genomic DNA was prepared as described in Appendix D. The DNA sequence of the
primers employed and the PCR conditions used were also described in Appendix D.
PCR amplification was performed in a model 1108 Tempcycler H (Coy Corporation,

Grass Lake, MI) or a Perkin & Elmer therrnocycler, using the following cycles: 1

 

 

 

 

155

Table 1: Bacterial isolates or DNA used in this study and
associated information.

 

MSU ID STRAIN HOST LOC- YEAR GROUP RACE STARCH PECT. CKTM Source

 

 

# 10 ATION Acrrv. or Ref.
CVP
666 Xv 36 PL A - - r JBJ
684 Xv 29 P OK 1990 A - - P cL8
685 Xv 31 P OK 1989 A - - P CLB
686 Xv 334 P CAR A 1 + - v JBJ
687 Xv 85 r PL A 1 - r JBJ
688 Xv 1 P PL A 1 - r JBJ
689 Xv 858 1 IX A 2 - - r JBJ
690 Xv 104 P ru A 3 1 - P JBJ
691 Xv91 P Tu A 3 - - v . JBJ
692 Xv 89 P ru A 3 - - P JBJ
694 91913 P our 1991 A - - P RB
697 Xv 110 P ru A 1 - - P JBJ
698 Xv 855 r nx A 2 - - r JBJ
699 Xv 856 r MX A 2 - - v JBJ
700 Xv 857 T AX A 2 - - 1 JBJ
701 Xv 859 1 ﬁx A 2 1 - v JBJ
702 Xv 18 1 FL A 1 - v JBJ
703 Xv 122 r ru A - - r JBJ
704 Xv 300 1 CAR A - - 1 JBJ
706 Xv 531 1 CAR A 2 - - r JBJ
739 Xv 597 P CAR A - - P JBJ
740 Xv 102 P ru A 1 - - P JBJ
741 Xv 63 P PL A 1 - P RES
742 Xv 92-17 P PL 1992 A 2 - - P RES
744 Xv 90-1P P GA 1990 A - - P RG
745 Xv 89-53P P GA 1989 A - - P RG
746 Xv 89-52P P GA 1989 A 1 P RG
747 Xv 88-45P P GA 1988 A - - P RG
748 Xv uc GA A - - 1 RG
837 SS-Pepper P-GH ONT 1992 A - - P DHAN
865 rs 8 1 our 1990 A - 1 P DHAN
866 rs 16 r our 1990 A - 1 P DHAN
867 rs 26 I out 1990 A - 1 P DHAN
868 rs 31 1 our 1990 A - - P DHAN
869 ts 35 1 our 1990 A 1 - P DHAN
878 Sp2-92 P-s GA 1993 A - - P Go
879 Sp66-92 P-s GA 1993 A - - P 60
880 sp124—92 P-s GA 1993 A 1 r P 60
882 sp133-92 P-s GA 1993 A - - P Go
883 Sp135-92 P-s GA 1993 A - - P G0
884 P93-DJA P-GH GA 1993 A 1 - P G0
886 Xv 18 (on) P on 1992 A 1 - - P S"
887 Xv 47 P on 1992 A 1 - - P 38
888 Xv 44 P on 1992 A 1 - - P sn
890 Xv 71 P OH 1992 A 1 - - P sn
908 Arcc 11633 P us 1947 A ~ - v Arcc
931 Xv 931 P-GH MI 1993 A . - P THIS STUDY
939 Xv 939 P MI 1993 A - ' P THIS STUDY
943 Xv 93-1 PL 1993 A 3 — - v RES
944 Xv 93-26 PL 1993 A 11 - - 7 RES
945 Xv 93-24 PL 1993 A 2 - - - RES
947 Xv 92-16 PL 1992 A 1 - - P RES
948 Xv 75-3 PL 1975 A 11 1 - v RES*
949 Xv 93-29 PL 1993 A 71 - - T RES
LMG 905 1982 A NA NA NA JS**
LMG 910 P nonocco1976 A NA NA NA JS**
LMG 929 P PL 1969 A NA NA NA JS**

   

 

Table 1 Continued...

156

 

MSU ID STRAIN
# ID

682

871

U!

ATCC 35937

ATCC 11551
ICBB 167
BA 27-1
BA 29-1
BV 5-3A
BV 6.1
BV 7.3A
BV 4.1
Xv 56
LNG 920
Xcv 981
Xcv 982

Xv 441

DC 92-6
DC 91-1

X-1
5-2-4
Xpel 942
Xp 805
JT1

JT4

ch 898
Pss 11

HOST

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NI
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YEAR GROUP RACE STARCH PECT.
AC

1987
1987

1992
1992
1992
1991
1992

1979
1955
1943

1993
1993

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AJ

AJ
THIS STUDY

 

157

TABLE 1 CONTINUED...
Footnotes to Table 1:

Geranium

Host: P = Pepper S = Seed geran
= arabidopsis

T = Tomato GH Greenhouse aradib =
Location: FL = Florida; GA = Georgia; OK =Oklahoma;
MI = Michigan; ONT = Ontario; NJ = New Jersey;

OH = Ohio; KS = Kansas; NZ = New Zealand;
Moro = Morocco; ARG = Argentina; TW = Taiwan;
CAR = Caribbean; MX = Mexico.

Race: (after Minsavage et al. 1990)

T1 = tomato race 1
1 = pepper race 1
2 = pepper race 2 (P2)
3 = pepper race 3 (P3)
T2 = tomato race 2 (after Wang etal. 1990)

not able to hydrolyze starch
hydrolyzed starch weakly
hydrolyzed starch extensively

Starch: (—)

+1+
u u u

v

Pectolytic activity on CVP medium: (-) = no activity
(+) = pectolytic
(:_|-_) = slight activity

CKTM: P = formed a clear ring, "pepper type"
T = formed opaque white precipitate "tomato type"
V = intermediate phenotype; - = no phenotype on CKTM

Source or reference: JBJ=J.Jones, Gulf Coast Research
Center, University of Florida; CLB=C. Bender, Department of
Plant Pathology, Oklahoma State UniverSity;.RB=R.Brammal,
Ontario Ministry of Agriculture and Food, Simcoe, Ontario;
RES=R. Stall, Department of Plant Pathology, UniverSity of
Florida; RG=R.Gitaitis, Department of Plant Pathology,
University of Georgia; DHAN=Dr. Dhanvantari, Agriculture
Canada, Harrow, Ontario; GO=G.O’Keefe, Georgia Dept.
Agriculture, Georgia; SM=S.Miller, Florida Dept. Agr. and
Consumer Serv., Gainesville; ATCC=American Type Culture
Collection; JS=J.Swings, Laboratorium voor Microbiologie,
Universiteit, Gent, Belgium; DC=D.Cuppels, Agriculturet
Canada, London, Ontario; KS=K.D:n:arI,J gsggggmsnt of Bo any
and Plant Patholo , Michigan S a e n .
MD=M.Daughtrey, Lghg Island Hort. Res. Lab.; LA=L.Afanador,
Department Crop and Soil Sciences, Michigan State 1 t
University; AJ=A.Jones, Department of Botany and FUSE-Plant
Pathology, Michigan State UniverSity; UT=J.Tsuql, f nces *
Research Laboratory, Michigan State UniverSity,.reuere l' u
Whalon et al. 1988; "'Vauterin et al. 1990,1991, Beau ie

‘et al. 1991 and/or Stall et al. 1994.

 

 

 

158

initial cycle at 95°C for 7 min; 30 cycles at 94°C for 1 min, annealing at 44°C, 52°C or
53°C for 1 min with REP, ERIC and BOX primers, respectively, and extension at 65°C
for 8 min with a single ﬁnal extension cycle at 65°C for 15 min, followed by a soak at
4°C. PCR mixtures were overlain with 25 ul of mineral oil (Sigma M3516). Each PCR
experiment included a control (no template DNA) and one or more controls with DNA
from another pathovar of X. campestris.

Approximately 6 ul of PCR generated DNA fragments were resolved by gel
electrophoreses at 4°C in 1.5% agarose gels in 0.75X or 0.5X TAE buffer at 5 V/cm.

Differences in ﬁngerprint patterns between groups were assessed visually.

PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION. All isolates
were evaluated by selected phenotypic tests, commonly used to characterize Xcv
strains (Gitaitis et al. 1987). Starch utilization on nutrient agar (Difco Laboratories,
Detroit, MI), amended with 1% soluble starch, was scored qualitatively as weak (_+_),
positive (+) or negative (-). After 2 to 3 days of bacterial growth, starch utilization
was considered negative if no clear zone formed when the medium was stained with
an iodine solution. Quantitative estimates of starch utilization were obtained for isolate
Xcv 736, ATCC 35937, Xv 75-3, and ATCC 11633 by dipping a 13 mm sterilized
ﬁlter disk in a bacterial suspension (108 cfu/rnl) and placing the disk on 15 ml of the
starch medium in the center of 9 cm petri dishes. Cleared zones in three samples of

each isolate were measured at 18 to 178 hrs after inoculation of the medium. The

 

linear portion of the data was expressed as starch hydrolysis in mm per hour based on
linear regression analysis.

Pectolytic activity was determined by growth on crystal violate pectate medium

 

 

 

 

159

(CVP) (Cuppels and Kelman 1974) and cellulolytic activity was determined on
carboxymethylcellulose medium (CMC) (Gitaitis et al. 1991). Colony characteristics
on a basal CKTM medium lacking antibiotics were recorded as described by Sijam et
al. (1992). Xcv isolates form a precipitate on CKTM and can therefore easily be
identiﬁed on this selective medium. Isolates from tomato develop opaque white halos
whereas isolates from pepper simply form a clear ring (Sijam et al. 1992).

Bacterial cultures were initiated on N YDA and cells were transferred to each
medium with an inoculating loop. All physiological and biochemical tests were
repeated 8 minimum of two times for each isolate. The quantitative experiment for

starch utilization was conducted once.

RESULTS

FOUR GENOTYPES ARE RESOLVED BY REP-, BOX- AND ERIC-PCR.

 

DNA ﬁngerprints were generated from total chromosomal DNA extracted from
over 80 isolates of Xcv originating from various parts of the world (Table 1). Primers
corresponding to REP, BOX and ERIC sequences, in combination with the polymerase
chain reaction, generated complex genomic ﬁngerprinting pattems from DNA of each
isolate of up to 20 or more PCR products, that ranged in size from approximately 0.2
to over 5 kb. Isolates were classiﬁed into 4 distinct groups based on these
ﬁngerprinting patterns (Figure 1). The REP- (lanes 1 to 6), BOX- (lanes 7 to 12), and
ERIC-PCR (lanes 13 to 18) experiments were equally effective in delineating the four

groups. Group A (lanes 1,7,13), Group B (lanes 2,8,14), Group C (lanes 3,9,15) and

 

Group D (lanes 4&5, 10&11, 16&17) included 56 (69%), 25 (31%), 1 (1.2%) and 2

   

 

 

 

 

160

(2.4%) of the isolates evaluated, respectively. Not one rep-PCR generated band
common to all groups was generated by the REP-, BOX-, or ERIC-PCR experiments.
With the ERIC—PCR experiment, one to 3 bands appeared to comigrate among isolates
classiﬁed as Group A or Group B. For example, isolate ATCC 11633 (Figure 1, lane
13) appeared to have 3 bands (highlighted by arrowheads in lane 13) that comigrated
with bands generated from chromosomal DNA of Xcv 736 (Figure 1, lane 14).
Sequencing or hybridization studies would need to be conducted to determine if the
comigrating bands are analogous portions of DNA in both the Group A and B isolates.
The rep-PCR experiments effectively differentiated 4 groups among strains
classiﬁed as Xanthomonas campestris pv. vesicatoria based on total chromosomal
ﬁngerprint patterns. Disparate ﬁngerprint proﬁles between the four groups, categorized
here as Group AB, C and D, suggest that the groups are genetically highly dissimilar.
Fingerprints generated from Xcv strains were unique as compared to ﬁngerprint
proﬁles generated from over 30 other xanthomonads (Appendix D and data not
shown), and numerous strains classiﬁed as Pseudomonas, Clavibacter, as well as
saprophytic bacteria associated with ﬁeld tomato plants, greenhouse tomato plants and

overwintered tomato debris (data not shown).

GENOTYPIC VARIATION WITHIN GROUP A AND B AS DETERMINED BY
BOX, REP-, AND ERIC-PCR.
In contrast to the near complete different ﬁngerprint patterns between groups,

rep-PCR ﬁngerprint proﬁles generated from DNA of isolates within each group were

hi8th similar (Figure 2 and 3).

 

 

 

 

 

 

 

 

161

 

FIGURE 1. Agarose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria using primers
corresponding to REP (lanes 1 to 6), BOX (lanes 7 to 12) and ERIC (lanes 13 to 18)
sequences. Six ul of PCR products were loaded in each lane. A typical Group A
pattern (lanes 1,7 and 13), Group B pattern (lanes 2,8 and 14), the Group C pattern
(lanes 3,9 and 15) and Group D pattern (lanes 4&5, 10&11 and 16&17) are displayed.
The right— and left-most lane contain DNA size markers (1 kb ladder, Gibco-BRL)
indicated in base pairs. Arrowheads identify similarities or differences among selected
isolates as outlined in the text. PCR bands were resolved on 1.5% agarose gels stained
with ethidium bromide.

 

 

162

BOX-PCR ERIC-PCR

REP-PCR

 

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163

FIGURE 2. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates of Xanthomonas campestris pv. vesicatoria Group A (Top) and
Group B (Bottom), using primers corresponding to BOX sequences. Other details are
as outlined in the legend of Figure 1.

 

 

 

 

 

 

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165

BOX-PCR differentiated l5 BOX-PCR ﬁngerprint types within Group A.
Pattems were highly similar (Figure 2 top) with differences limited to the presence or
absence of 1 to 3 bands, as compared to the predominant pattern highlighted by
ATTCC 11633 (lane 1). For example, LMG905, T835, T88 and Xv93-29 (lanes 2 to
5) each yielded an extra single band of approximately 700 bp (opposing arrows in
lanes 2 and 5). Likewise, the isolates Xcv939, Xcv931, Sp135, Sp133, Xv89, Xv334
and leO4 (lanes 10 to 16) yielded a polymorphic band about 950 bp in size
(opposing arrows in lanes 10 and 16). However, each of the latter isolates were not
completely similar. For example, isolate Xv334 did not yield two bands in the 3 kb
range and possessed a distinct polymorphism at 600 bp. Examples of other
polymorphisms generated from other isolates are highlighted by arrowheads.

BOX-PCR delineated 5 patterns among strains classiﬁed as Group B (Figure 2
bottom, polymorphisms highlighted by arrowheads). The distinct BOX-PCR
ﬁngerprinting patterns could not be associated with geographic region. For example
TSl originated in Ontario in 1979 and had a BOX-PCR ﬁngerprint indistinguishable
from the two Oklahoma isolates (leO and Xv15,1987) and two isolates from
Michigan (Xcv859 from 1991 and Xcv736 from 1992; see Figure 28, lanes 1 to 5).
Likewise, Xcv 981 and 982, isolated from different Michigan ﬁelds of processing
tomato in 1993, had BOX-PCR ﬁngerprint patterns identical to BV6-1 and BV4-1
isolated in Argentina. Xv56 was also similar to the latter 4 isolates. The date of
isolation also did not appear to affect the rep-PCR fingerprint proﬁles of Xcv strains.
ATCC 35934 is the pathovar reference strain isolated from New Zealand in 1955 and
could not be distinguished from 5 strains (DC92-13, DC92-21, DC92~23, CC164 and

CC195) isolated from independent epidemics in Southwest Ontario in 1992 (see lanes

 

 

 

 

 

 

 

 

166
6 to 14). BA27-1 and BA29-l (lanes 20 and 21) were also indistinguishable from each
other. BV5-3A generated a unique proﬁle (lane 22).

ERIC-PCR differentiated 13 ﬁngerprint types within Group A. As with BOX-
PCR, patterns were highly similar with differences limited to the presence or absence
of 1 to 3 bands (Figure 3). Arrowheads highlight several of the unique characters that
were scored. For example, le8, Xv531, leO4 and Xv334 each lacked a prominent
1.9 Kb band (lanes 10 to 13 highlighted by opposing arrows). Xv334 (lane 13). was the
least similar to the other memebers of Group A with regard to ﬁngerprint pattems
generated.

ERIC-P CR ﬁngerprinting lead to the inclusion of the Group B strains into the
exact same clusters as BOX-PCR. The 5 unique banding patterns displayed abundant
common bands (Figure 4A, lanes 1 to 5) but numerous additional bands generated
strain speciﬁc ﬁngerprint proﬁles.

The relatively low total number of bands generated by REP-PCR rendered the
REP primer set the least useful for discriminating among strains within Group A. All
isolates within Group A generated REP-PCR ﬁngerprints identical to ATCC11633 (see
Figure 1, lane 1) with two exceptions (Figure 5). Xv334 yielded two additional
prominent bands and a second collection of isolates (SP133, Sp135, Xv18-OH,
Xcv931, Xcv939 and Xv89) yielded a single additional band. REP-PCR differentiated
Group B strains similar to BOX- and ERIC-PCR (Figure 4B). However, Xv56 yielded
an additional band as compared to BV4-1 and BV6—1 (data not shown). —

The combined data (BOX, ERIC and REP) appeared to provide a more detailed
assessment of the chromosomal structure and strain diversity as compared to data

generated by one primer set alone. Strain speciﬁc rep-PCR ﬁngerprint patterns were

 

 

 

 

 

 

167

 

FIGURE 3. Agarose gel electrophoresis of ﬁngerprint patterns obtained from genomic

DNA from isolates of Xanthomonas campestris pv. vesicatoria Group A using primers
corresponding to ERIC sequences. Other details are as outlined in the legend of Figure
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169

FIGURE 4. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
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(A) and REP (B) primers. Other details are as outlined in the legend of Figure 1.

 

 

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171

FIGURE 5. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
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corresponding to REP sequences. Other details are as outlined in the legend of Figure
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172

 

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173

much more distinct within Group B. Group B appears to be comprised of a more

heterogenous group of strains than Group A.

Genotype of Group D Strains. The isolates classiﬁed as Group D (Figure 1, lanes
4&5, 108811 and 16&17) appeared to have several prominent bands of parallel
mobility, but numerous additional bands were ampliﬁed to generate strain speciﬁc
proﬁles. We compared the rep-PCR ﬁngerprint proﬁle of these strains to those
generated from numerous other xanthomonad pathovars during our studies. We found
that the two Group D strains shared several bands in common with isolates of
Xanthomonas campestris pv. campestris (ch) (Figure 6). Several rep-PCR generated
bands of apparently analogous DNA products between Group D and ch strains
comigrated after BOX-PCR analysis (highlighted by arrowheads, Figure 6, lanes 1-4).
A representative isolate of Xcv (Xv29, lane 5) did not appear to share more than 1 or
2 bands with Group D or ch strains. Three REP-PCR bands between 3 to 3.9 kb
appeared to be common between the 2 Group D strains and two ch strains, ATCC
33913 and ch898 and also representative isolates of Xcv (data not shown). An
additional REP-PCR band of 800 bp was common to the Group D and ch strains
(data not shown). The BOX—PCR experiments, however, provided the strongest
evidence of a possible close genetic relationship between the Group D and ch

strains.

Reproducibility of ﬁngerprints. The similar rep-PCR ﬁngerprint proﬁles generated
from isolates separated by a 40-50 year period attest to the reproducibility of

fingerprints generated by REP-, ERIC- and BOX-PCR. In addition, the pathovar

 

 

 

174

FIGURE 6. A garose gel electrophoresis of ﬁngerprint patterns obtained from genomic
DNA from isolates classiﬁed as Xanthomonas campestris pv. vesicatoria Group D
(lanes 1&2) as compared to patterns generated from representative Group A isolates
(lane 5) and isolates of Xanthomonas campestris pv. campestris (lanes 3&4) using
BOX primers. Other details are as outlined in the legend of Figure 1.

 

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176
reference strain was received as a culture or DNA from 3 independent sources (ATCC;
J. Jones, Florida; and J. Swings, Belgium). Each primer set yielded identical proﬁles
from DNA for each of the 3 samples. Likewise, duplicate samples of other strains
were received over time and/or independently prepared and analyzed to yield identical
ﬁngerprints (data not shown). Dispensing cells directly into the PCR tubes from liquid
or solid medium cultures also yielded ﬁngerprint pattems identical to patterns
generated from isolated DNA (data not shown). Finally, unique bands, such as those
highlighted by arrowheads in Figure 2A, could be reproduced by independent rep—PCR
experiments and independent analysis of aliquots of the same PCR mixtures on

agarose gels.

PHENOTYPIC CHARACTERISTICS WITHIN GROUP A, B, C, AND D.
A Because the ﬁngefprint proﬁles between the four different groups were so distinct, we
conducted several phenotypic tests to determine if speciﬁc phenotypes were associated
with each group. Starch utilization, pectolytic activity and cellulolytic activity are
common tests to differentiate Xcv from other pathogens and saprophytes in Georgia
and Florida (Gitaitis et al. 1987). CKTM is a medium selective for the isolation of
Xcv and is able to differentiate tomato and pepper strains (Sijam et al. 1991, 1992).

GROUP A Phenotype: Isolates classiﬁed within Group A were obtained from

various parts of the world, were commonly isolated from tomato or pepper and were
non-pectolytic (Table 1). Seventy- seven percent of Group A isolates were starch
negative, 21% hydrolyzed starch weakly and one isolate (Xv334) was starch positive

(Table 1).

Within Group A, a total of 18 and 35 isolates originated from tomato and

 

 

 

 

 

 

177
pepper, respectively (Table 1). All Group A isolates tested, except Xv93-24, formed a
precipitate on CKTM medium. Although the biochemical and genetic basis for the
differential reaction on CKTM is not known, 82% of strains isolated from pepper
formed a pepper-type precipitate as described by Sijam et a1. (1992). Fifty percent of
the strains isolated from tomato did not form a distinct tomato-type precipitate. Five of
these 9 isolates formed a pepper precipitate on CKTM medium and came from
Ontario (Ts8,Tsl6,TsZ6,Ts31,Ts35). Each isolate was obtained from a different ﬁeld
but all in the same general geographic region (Southwest, Ontario) and in 1990 (Dr.
Dhanvantari, personal communication).

GROUP B Phenotype: Isolates classiﬁed as group B also came from various
parts of the world, including South America, New Zealand, Canada (ON), and the
United States (MI & OK) (Table 1). All isolates evaluated and classified within Group
B were able to hydrolyze starch, demonstrated pectolytic activity on CVP medium and
with one exception (BA27-1) originated from tomato (Table 1).

Ninety one percent of the Group B strains did not form a halo on CKTM
medium (Table 1). ICBB167 formed a very subtle clear ring within 3 days and BV7-
3A and leO had a light white halo after 4 and 6 days, respectively.

GROUP C and D Phenotyp_e: Strain Xv441, the single isolate classiﬁed as
Group C, originated from tomato and was negative for both amylolytic and pectolytic
activity (Table 1). Xv441 formed a subtle ring on CKTM similar to strain ICBBl67 of
Group B (Table l). The two Group D isolates, DC91-1 and DC92-6 from Ontario,
originated from tomato greenhouse transplants (D. Cuppels, personal communication),

were starch and pectolytic positive and formed a distinct tomato-type halo on CKTM

media (Table l).

 

 

 

 

 

 

 

178

All Xcv strains tested demonstrated cellulolytic activity (data not shown).
Isolates of Xanthomonas campestris pv. campestris were CMC positive, positive for
starch hydrolysis and pectolytic activity and formed a tomato-type precipitate or no
precipitate on CKTM (Table 1). Other pathovars of Xc were CMC positive and were

negative or positive for starch hydrolysis and pectolytic activity (Table 1).

 

Pseudomonas isolates effectively functioned as controls and scored negatively on CVP,

starch medium (Table 1) and CMC.

Quantitative assessment of starch hydrolysis: Several isolates were found to
hydrolyze starch weakly (Table 1) and this is a common description for strains
classiﬁed as Xanthomonas campestris pv. vesicatoria (Dye 1962; Dye et al. 1964;
Gardner and Kendrick 1921). Therefore an assay was performed to determine the
quantitative differences between strains that hydrolyze starch weakly as compared to
those with strong hydrolytic activity. Isolate ATCC 35937 (pathovar reference strain)
and Xcv 736 demonstrated identical amylolytic activity (Figure 7 ). Both isolates
formed clear zones within 18 hours and the zones expanded at a rate of O. 12 mm per
hr between hrs 56 to 180 (Figure 7). A representative starch negative isolate, ATCC
11633, did not form a zone even after 7 days. Xv75-3, a weak hydrolyzer of starch,
did not form a distinct zone until after 56 hrs and the subsequent rate of zone

eXpansion was 0.07 mm per hr thereafter.

 

Distribution of Races. Each described known race sensu Minsavage et al. (1990),

 

including tomato race 1 (T1 of the Xch group), pepper race 1 (0f the XCVP group),

pepper race 2 (of the XchT group) and pepper race 3 (of the XchT group) had

   

 

 

 

179

 

FIGURE 7: Increase (mm) in the radius of cleared zones indicating starch hydrolysis
by selected isolates of Xanthomonas campestris pv. vesicatoria.

 

 

 

 

 

mm301

 

180

 

 

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181
genomic rep-PCR ﬁngerprints characteristic of Group A (Figures 2A and 3A). Race
designation of isolates within Group A could not be correlated to total chromosomal
ﬁngerprint patterns as determined by rep-PCR. Isolate Xv441 from the Caribbean
Islands has been classiﬁed as race 1 (personal communication, H. Bouzar and J. Jones,

Gulf Coast Research Center, Florida) and was the single member of Group C.

DISCUSSION

The precise function of REP, BOX and ERIC sequences is not known (Lupski
and Weinstock 1992) but these repetitive sequences were exploited via PCR to rapidly
assess the genetic diversity of strains classiﬁed as Xanthomonas campestris pv.
vesicatoria. Based on this study, strains classiﬁed as Xanthomonas campestris pv.
vesicatoria clearly fell into 4 completely different groups, designated A, B, C and D,
Isolates appear to be highly homogenous within Group A and isolates within Group B
are more genetically diverse.

We noted polymorphisms among Group A, B and D isolates by REP- BOX-
and/or ERIC-PCR. Polymorphisms were sirnple (with differences limited to 1 to 3
DNA bands with any given primer set) within Group A, with the exception of Xv334.
This isolate was shown to be polymorphic using all 3 primer sets. Xv334 was also
physiologically atypical having starch hydrolytic activity. In contrast, in our limited
sample of Group B, 5 distinct patterns, or lineages, could be elucidated by each primer
set. Stall et al. (1993) have also concluded that Group B appears to comprise a more
heterogenous collection of strains as compared to Group A.

Despite the heterogeneity of patterns within Group B, it does not appear to be

 

 

 

 

 

 

 

182

possible to follow a speciﬁc strain in epidemiological studies using the rep-PCR. For
example, Xcv859 was isolated in 1 of 112 plots during a large integrated disease
management study in Michigan during the 1991 season (Chapter 2). In 1992, Xcv736
was isolated from the exact same plot and yielded a ﬁngerprint identical to Xcv859,
suggesting the strain had overwintered under the minimum tillage conditions
employed. However, isolates from Ontario (T81) and Oklahoma (leO and leS) also

had identical ﬁngerprint patterns (Figure 28 and 3). Although unlikely in this

 

particular ﬁeld study, the possibility that Xcv736 was reintroduced as seed-borne
inoculum could not be ruled out.

Xcv has been described as a pathovar comprised of diverse strains (Doolittle
and Crossan 1959, Dye 1962, Dye et al. 1964, Jones et al. 1993a, Minsavage et al.
1990, Klement 1959, Sutic 1959, Vauterin et al. 1990, 1991, Wang et al. 1990,
Whalen et al. 1988). This report highlights the complexity of the observed diversity
based on the ability of different isolates to hydrolyse starch, their pectolytic activity,
reaction on CKTM medium, host of origin and pathogenicity. However, in this study
we have been able to categorize such diversity within a useful genotypic framework as
determined by rep-PCR. For example, Xcv has been described with the ability

(Gardner and Kendrick 1921), inability (Gitaitis et al. 1987) and variable ability

 

(Burkholder and Li 1941; Dye et al. 1964) to hydrolyse starch and demonstrate
pectolytic activity (Beaulieu et al. 1991). Our work demonstrates that amylolytic and
pectolytic activities are highly associated with speciﬁc groups. Over 96% of the
isolates evaluated in this study were classiﬁed as Group A or Group B and the isolates
in each group were predominantly amylolytic/pectolytic minus or amylolytic/pectolytic

P1118, respectively.

 

 

 

183

Vauterin et al. (1990, 1991) have classiﬁed Xcv strains into two sub-pathovar
categories, subgroup A and B, based on fatty acid proﬁle analysis, DNA homology
studies and SDS-PAGE Electrophoresis. Likewise, Jones et al. (1993) and Stall et al.
(1993) have classiﬁed Xcv strains at the subpathovar level as Strain A and Strain B
based on polyphasic criteria. We obtained representative samples from both research
groups and learned that subgroup A and subgroup B is synonymous with Strain A and
Strain B, respectively. Prior to a comparative analysis of the alternative subpathovar
classiﬁcation systems, we had classiﬁed the various groups discerned by rep-PCR as
group I, II, III and IV (Int. Congress Plant Pathology, Montreal, 1993). However, to
avoid confusion and because current use of A and B is in agreement, we also used
alphabetical letters in this chapter to name the groups.

Harmonizing the two subpathovar groupings allowed a more comprehensive
understanding of the diverse attributes associated with group A and B. For example,
Vauterin et al. (1990, 1991) have shown A and B strains belong to distinct DNA
homology groups and have different SDS-PAGE proﬁles. Isolate LMG 920 and the
pathovar reference strain (ATCC 35937) have been classiﬁed into separate subgroups
based on SDS-PAGE proﬁles (Vauterin et al. 1991) but could not be distinguished by
rep-PCR (Figure 2, bottom).

Stall et al. (1993) have demonstrated that Group A and Group B can also be
differentiated by monoclonal antibodies and restriction endonuclease analysis among
other methods tested. They also corroborate our observations of pectolytic activity and
starch hydrolysis associated within Group B but not Group A.

The REP-, BOX-, and ERIC-PCR ampliﬁed identical proﬁles from genomic

DNA of the Group B strains BA27-1 and BA29-1 and this proﬁle differed from BV5-

 

 

 

 

 

184
3A and Xv56. Stall et al. (1993) have also used these isolates in their study and
showed monoclonal antibodies could distinguished BA27-1/BA 29-1 as compared to
BV5-3A and Xv56. Based on restriction digest analysis Stall et al. (1993) also noted
that BV27-1 and BV29-1 have a very small genetic distance whereas Xv56 and BV5—
3A have a greater genetic distance, further corroborating the results of this study.

The fact that strains classiﬁed as Group A and B are so dissimilar suggests that
pathogenicity for tomatoes occurred through convergent evolution and that the
population structure of Xcv is polyphyletic. The relative contribution of
recombinational convergence or mutational convergence (Selander and Musser 1990)
is not known. If recombinational convergence presides, then limited host-speciﬁc
virulence genes (Swarup et al. 1992; Waney et al. 1991) may associate with diverse
genomic backgrounds to give rise to new genotypes pathogenic for tomato and/or
pepper but with an unaltered host range. For example, Group D and Group A isolates
may have the same host-speciﬁc gene in a different genetic background. In such cases,
the possibility of developing durable genetic resistance appears to hold more promise
than if pathogenicity occurs through mutational convergence i.e. perhaps through the
mutation of an avirulence factor (Kearney et al. 1988). In the latter case, rates of
mutation and selective pressure may always favor parasitism.

The importance of Group C and D genotypes and the presence of other
genotypes pathogenic to tomato and/or pepper is not known. The single group C strain
(Xv441) was discovered in the Caribbean region and has been noted to be unique
based on polyphasic phenotypic experiments (H. Bouzar and J .B. Jones, University of

Florida, personal communication). The Group D isolate DC91-1 is a highly virulent

and destructive pathogen based on our pathogenicity tests (data not shown) and

 

 

 

185

economic damage observed in a commercial greenhouse (D. Cuppels, personal
communication, Agriculture Canada, London, Ontario). DC92-6 was recovered from
tomato seedlings in the greenhouse, shares numerous comigrating bands as DC91-1 but
does not appear to be highly virulent to tomato (data not shown). Based on common
bands between these Group D strains and isolates representative of Xanthomonas
campestris pv. campestris (ch), our data suggests the Group D strains may have
originated from or have a common ancestry as ch. Additional sampling and
evaluation may in fact reveal other genotypes able to incite bacterial spot of tomato.
Assessing and monitoring clonal population structures may provide a means for
determining the potential of new genotypes to emerge and become prominent through
periodic selection and extinction (Levin 1981) forces and provide some insight into the
evolutionary dynamics of plant pathogenesis (Selander and Musser 1990). The
detection of these genotypically distinct strains (DC91-1 and DC92-6) invites
numerous questions on the origin of these strains, the genetic differences between the
highly virulent strain (DC91-1) and the less virulent strain (DC92-6), and the potential
of the highly virulent strain to become a predominant clone.

The importance of discerning genetic diversity is highlighted by our ﬁndings
that Group B strains comprise an important component of the tomato spot complex in
the northcentral production region. During the initial phase of our study, most strains
we evaluated belonged to Group A. Local Michigan isolates obtained from infected
tomatoes yielded a totally dissimilar fingerprint proﬁle via rep-PCR when compared to
Group A strains and we thought we may have isolated saprophytic xanthomonads
(Gitaitis et al. 1987). However, when we obtained the pathovar reference strain (ATCC

35937) and numerous other isolates from Ontario, the latter new isolates had a similar

 

 

 

 

 

186
ﬁngerprint proﬁle as our Michigan isolates and prompted us to determine their
amylolytic and pectolytic activity. Our results indicate Group B strains are more
widely distributed than previously thought. Most work with Xcv has been conducted in
Florida and Georgia where the majority of isolates are diagnostically unable to
hydrolyze starch and are non-pectolytic (Beaulieu et al. 1991; Gitaitis et al. 1987; Stall
et al. 1993). Beaulieu et al. (1991) concluded pectolytic activity was correlated with
the geographical origin of isolation, since 90% of isolates from Argentina had
pectolytic activity (including isolates used in our study such as Xv 56, BV5-3a, and
BA27-1, i.e. Group B) compared to 0.003% from the United States. However, strains
obtained from numerous epidemics in Ontario (DC92-13, DC92-21, DC92-23, T81,
CC164#3 and CC195#1), Michigan (Xcv859, 736, 981, 982), Indiana (ATCC 11551)
and Oklahoma (Xv 10 and Xv 15), all belong to Group B.

Although it is difﬁcult to ascertain, low levels of Xcv detected on tomato seed
as compared to pepper seed may be a function of detection protocols used. In the
future, protocols should be designed to detect, at the minimum, Group A and B
isolates. Likewise, plant breeding programs will need to include representative strains

of each group. Diagnostic and breeding programs need to be integrated into the overall

 

tomato production system and are knowledge based and dependent on the tomato
industry as a whole, as compared to ﬁeld level integrated management strategies.
Intensive research and development is required to ensure Xcv is not a problem on
farms. Once Xcv becomes established in a ﬁeld, there is little a grower can do to
control the epidemic in susceptible cultivars.

In conclusion, elucidating the genotypic structure in natural populations of

strains classiﬁed as Xcv provided a framework for mapping the diversity of

 

 

 

187

phenotypic traits including virulence and pathogenicity. Knowledge of the population
structure of Xcv should aid in the selection of representative isolates for taxonomic
analyses, for evolutionary, ecological, epidemiological studies, and for devising
integrated disease management of bacterial spot of tomato such as diagnostic,

detection and plant breeding programs.

 

 

 

 

 

 

188
LITERATURE CITED

Beaulieu, C.B., G.M. Minsavage, B.C. Canteros, and RE. Stall. 1991. Biochemical
and genetic analysis of a pectate lyase gene from Xanthomonas campestris pv.
vesicatoria. Mol. Plant-Microbe Interact. 4:446—451.

Burkholder, W.H. and CC. Li. 1940. Variations in Phytomonas vesicatoria.
Phytopathology 31:753-755.

Cuppels, D., and A. Kelman. 1974. Evaluation of selective media for isolation of soft-
rot bacteria from soil and plant tissue. Phytopathology 64:468-47 5 .

de Bruijn, F.J. 1992. Use of repetitive (repetitive extragenic palindromic and
enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain
reaction to ﬁngerprint the genomes of Rhizobium meliloti isolates and other soil
bacteria. Appl. Eviron. Microbiol. 58:2180-2187.

Doidge, EM. 1921. A tomato canker. Ann. Appl. Biol. 7:407-430.

Doolittle, S.P. and CF. Crossan. 1959. Strains of Xanthomonas vesicatoria (Doidge)
Dowson differing in virulence on tomato and pepper. Plant Dis. Rptr. 43:1153

Dye, D.W. 1962. The inadequacy of the usual determinative tests for the identiﬁcation
of Xanthomonas spp. N.Z. J. Sci. 5:393-416.

Dye, D.W., M.P. Starr, and H. Stolp. 1964. Taxonomic clariﬁcation of Xanthomonas
vesicatoria based upon host speciﬁcity, bacteriophage sensitivity and cultural
characteristics. Phytopathol. Z. 51:394-407.

Gardner, M.W. and J.B. Kendrick. 1921. Bacterial spot of tomato. J. Agr. Res. 21:123-
156.

Gilson, E., J .M. Clement, D. Brutlag, and M. Hofnung. 1984. A family of dispersed
repetitive extra genic palindromic DNA sequences in E. coli. The EMBO J.
3:1417—1421.

Gitaitis, R.D., M.J. Sasser, R.W. Beaver, -T.B. McIrmes, and RE. Stall. 1987.
Pectolytic xanthomonads in mixed infections with Pseudomonas syringae pv. syringae,
P. syringae pv. tomato, and Xanthomonas campestris pv. vesicatoria in tomato and
pepper plants. Phytopathology 77:611-615.

Gitaitis, R.D., C.J. Chang, K. Sijam and CC. Dowler. 1991. A differential medium for
semiselective isolation of Xanthomonas campestris pv. vesicatoria and other
cellulolytic xanthomonads from various natural sources. Plant. Dis. 75 :1274-1278.

Goode, M.J., and M. Sasser. 1980. Prevention~ The key to controlling bacterial spot
and bacterial speck of tomato. Plant Dis. 64:831-834.

 

 

 

 

 

189

Hausbeck, MK. and 1.]. Kusnier III. 1993. Evaluation of flmgicides for the control of
bacterial spot of peppers. Fungicide and Nematicide Tests 48:143.

Hayward, A.C., and J .M. Waterston. 1964. Descriptions of Pathogenic Fungi and
Bacteria. No. 20, ”Xanthomona: vesicatoria”, Commonw. Mycol. Inst, Kew, Surrey,
England .

Higgins, BB. 1922. The bacterial spot of pepper. Phytopathology 12:501—516.

Higgins, C.F., G.F.-L. Ames, W.M. Barnes, J.M. Clement, and M. Hofnung. 1982. A
novel intercistronic regulatory element of prokaryotic operons. Nature 298:760-762.

Hulton, C.S.J., C.F. Higgins, and RM. Sharp. 1991. ERIC sequences: a novel family
of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and
other enterobacteria. Mol. Microbiol. 5:825-834.

Jones, J.B., J.P. Jones, R.E. Stall, and T.A. Zitter. 1991. Compendium of tomato
diseases. APS Press, St. Paul, MN, p.73.

Jones, J.B., S.M. McCarter, and RD. Gitaitis. 1981. Association of Pseudomonas
syringae pv. syringae with a leaf spot disease of tomato transplants in southern
Georgia. Phytopathology 71:1281-1285.

Jones, J.B., G.V. Minsavage, RE. Stall, R.O. Kelly, and H. Bouzar. 1993. Genetic
analysis of a DNA region involved in the expression of two epitopes associated with

lipopolysaccharide in Xanthomonas campestris pv. vesicatoria. Phytopathology
83:551-556.

Kearney, 8., RC. Ronald, D. Dahlbeck, and B.J. Staskawicz. 1988. Molecular basis
for evasion of plant host defence in bacterial spot disease of pepper. Nature 332:541-
543.

Klement, Z. 1959. Some new speciﬁc bacteriophages for plant pathogenic
Xanthomonas spp. Nature 184:1248-1249.

Koeuth, T., J. Versalovic, and J .R. Lupski. 1993. Differential subsequence
conservation supports the mosaic nature of interspersed repetitive BOX elements in
bacteria. Submitted.

Levin, BR. 1981. Periodic selection, infectious gene exchange and the genetic
structure of E. coli populations. Genetics 9911-23.

LUpski, J .R., and GM. Weinstock. 1992. Short, interspersed repetitive DNA sequences
in prokaryotic genomes. J. Bacteriol. 174:4525-4529.

Martin, B., O. Humbert, M. Camara, E. Guenzi, J. Walker, T. Mitchell, P. Andrew, M.
Prudhomme, G. Alloing, R. Hakenbeck, D.A. Morrison, G.J. Boulnois and J.-P.

 

 

 

 

 

190

Claverys. 1992. A highly conserved repeated DNA element located in the chromosome
of Streptococcus pneumoniae. Nucleic Acids Res. 20:3479-3483.

Minsavage, G.V., B.I. Canteros, and RE. Stall. 1990. Plasmid-mediated resistance to
streptomycin in Xanthomonas campestris pv. vesicatoria. Phytopathology 80:719-723.

Minsavage, G.V., D. Dahlbeck, M.C. Whalen, B. Kearney, U. Bonas, B.J. Stakawicz
and RE. Stall. 1990. Gene-for-gene relationships specifying disease resistance in
Xanthomonas campestris pv. vesicatoria-pepper interactions. Mol. Plant-Microbe
Interact. 3:41-47.

Pohronezny, K., and RB. Volin. 1983. The effect of bacterial spot on yield and
quality of fresh market tomatoes. HortScience 18:69-70.

Scott, J.W., G.C. Somodi, and J.B. Jones. 1989. Resistance to bacterial spot fruit
infection in tomato. HortScience 24:825-827.

Selander, R.K., and J .M. Musser. 1990. Population genetics of bacterial pathogenesis,
p. 11-36. In B.H. Iglewski and V.L. Clark (ed), Molecular basis of bacterial
pathogenesis.

Sharples, G.J., and R.G. Lloyd. 1990. A novel repeated DNA sequence located in the
intergenic regions of bacterial chromosomes. Nucleic. Acids Res. 18:6503-6508.

Sherf, AF. and A.A. MacNab. 1986. Vegetable diseases and their control. 2nd edition,
John Wiley & Sons, NY. 728 pp.

Sijam, K., C]. Chang, and RD. Gitaitis. 1992. A medium for differentiating tomato

and pepper strains of Xanthomonas campestris pv. vesicatoria. Can. J. Plant Pathol.
14:182-184. '

Stall, R.E., D.C. Loschke, and J.B. Jones. 1986. Linkage of copper resistance and
avirulence loci on a self-transmissible plasmid in Xanthomonas campestris pv.
vesicatoria. Phytopathology 76:240-243.

Stall, R.E., C. Beaulieu, D. Egel, N.C. Hodge, R.P. Leite, G.V. Minsavage, H. Bouzar,
J.B. Jones, A.M. Alvarez, and A.A. Benedict. 1993. Two genetically diverse groups of
strains are included in Xanthomonas campestris pv. vesicatoria. Int. J. System.
Bacteriol. In press.

Stern, M.J., G.F.-L. Ames, N.H. Smith, E.C. Robinson and CF. Higgins. 1984.
Repetitive extra genie palindromic sequences: a major component of the bacterial
genome. Cell 37:1015-1026.

Swarup, 8., Y. Yang, M.T. Kingsley, and D.W. Gabriel. 1992. An Xanthomonas citrz'
pathogenicity gene, pthA, pleitr0pically encodes gratuitous avirulence on nonhosts.
Mol. Plant-Microbe Interact. 5:204-213.

 

 

 

 

 

191

Sutic, D. 1957. Tomato bacteriosis. Inst. Zasht. Bilja, Beograd. 6, 65 pp. Abstr. in
Rev. Appl. Mycol 36:734.

Vauterin, L., J. Swings, K. Kersters, M. Gilles, T.W. Mew, MN. Schroth, N.J.
Palleroni, D.C. Hildebrand, D.E. Stead, E.L. Civerolo, A.C. Hayward, H. Maraite, RE.
Stall, A.K. Vidaver, and J .F. Bradbury. 1990. Toward an improved taxonomy of
Xanthomonas. Int. J. Syst. Bacteriol. 40:312-316.

Vauterin, L., J. Swings, and K. Kersters. 1991. Grouping of Xanthomonas campestris
pathovars by SDS-PAGE of proteins. J. Gen. Microbiol. 137:1677-1687.

Versalovic, J., T. Koeuth, and J .R. Lupski. 1991. Distribution of repetitive DNA
sequences in eubacteria and application to ﬁngerprinting of bacterial genomes. Nucleic
Acids Res. 19:6823-6831.

Waney, V.R., M.T. Kingsley, and D.W. Gabriel. 1991. Xanthomonas campestris pv.
translucens genes determining host-speciﬁc virulence and general virulence on cereals
identiﬁed by Tn5-gusA insertion mutagenesis. Mol. Plant-Microbe Interact. 4:623-627.

Wang, J.F., J.B. Jones, J.W. Scott, and RE. Stall. 1990. A new race of the tomato

group of strains of Xanthomonas cam pestris pv. vesicatoria. Phytopathology
80: 1070(abstr).

Whalen, M.C., RE. Stall, and B.J. Staskawicz. 1988. Characterization of a gene from
a tomato pathogen determining hypersensitive resistance in non-host species and
genetic analysis of this resistance in bean. Proc. Natl. Acad. Sci. USA 85:6743-6747.

 

 

 

 

CHAPTER V

CONCLUSIONS AND FUTURE RESEARCH

CONCLUSIONS

Over 70 to 100 years of research, embracing hundreds of independent studies,
document the epidemiology, etiology, and pathology of fungi and bacteria pathogenic
to tomato. An historical emphasis on chemical based disease control has left a void in
scientiﬁc and lay literature concerning the potential and implementation of alternative
forms of disease control. The relatively cheap and effective use of chemicals has
enabled the tomato industry to make signiﬁcant advancements in productivity (yield
per hectare) without relying on genetic resistance, cultural practices, or an
understanding of pathogen diversity for disease control. However, in the face of
declining chemical options, alternative disease management options are urgently
needed. In the case of genetic resistance, the limitation is not simply a lack of useful
germplasm, but is also likely due to a lack of emphasis. Likewise, cultural practices
must now be considered more seriously as a component of integrated disease
management strategies.

The knowledge base is limited concerning the effects and potential of rotation
(length and crops), cover and green manure crops, reduced tillage, and other
agronomic activities on management of disease in tomato production systems.
Likewise, limited information is available concerning the genetic diversity of tomato
pathogens. Advancements in cultural and bio-technologies are becoming available and

the balance of genetic, cultural and biotechnological inputs for integrated disease

192

 

 

 

 

193
management will be influenced by the pathosystem.

Fundamentally, as highlighted in this thesis, the most effective integration of
disease management strategies is based on the divergent mechanisms by which the
fungi and bacteria become epidemiological problems. The fungi are indigenous to the
region and appear to reside in the agro-ecosystem at inoculum levels high enough to
limit fruit quality even in ﬁelds with no history of tomato. Ultimately, disease
management of the fungal fruit—foliar pathosystem is dependent on farm level
decisions and inputs. Based on this thesis, it is possible to minimize the number
frmgicide applications required for the control of early blight, anthracnose, and soil rot
of fresh market and processing tomato through the use of tillage, a green manure crop,
and weather timed fungicide sprays even within a biennial (tomato/cucumber)
conservation tillage production system. The beneﬁts of conservation tillage, cover
crops and reduced fungicide input can be integrated without compromising fruit
quality and yield.

The bacteria are generally considered introduced problems and grower attempts
to control epidemics in susceptible cultivars have marginal impacts, especially during
climatic conditions that favor disease progress. One of the most prominent Michigan
growers of tomato plug transplants testiﬁed that the bacterial diseases appear to
become a problem despite any and all efforts on his part to try and limit them (M.
Hausbeck, personal communication). ”Prevention is the key” (Goode and Sasser 1980)
to integrated management and can only come about through industry dependent
activity such as private and public research and certiﬁcation protocols. Bacterial
canker, bacterial speck and bacterial spot were encountered at economic levels in plots

of the ﬁeld experiments outlined in this thesis (Appendix B) but the spot organism

 

 

 

 

 

 

 

194
was chosen as a model system for assessing genotypic diversity. Rep-PCR applied to
Xcv uncovered a population diversity that has direct consequence for disease
management. Starch positive and/or pectolytic strains comprise an important
component of the spot problem in the north-central production region. Attempts to
develop genetic resistance, assay seed lots for seed-borne inoculum, and the

development of diagnostic/identiﬁcation protocols must consider such diversity. This

 

requires high inputs in terms of research and development before ”the seed is planted”.

 

 

 

 

195

FUTURE RESEARCH

Integrated disease management of the fungal disease pathosystem is likely to be
advanced in several ways. Considerable beneﬁt was obtained with zone tillage (ZT) in
plots with no recent history of tomato. The length of rotation and type of crop may
impact initial inoculum and impact the ZT advantage each year. Although rotation is
routinely recommended, limited quantitative information appears to be available with
regard to inoculum levels and subsequent disease progress. Long term studies
incorporating various rotation treatments may provide a knowledge base for future
recommendations.

Management of the fungal pathosystem is likely to beneﬁt most from genetic
resistance. Resistance to early blight, anthracnose and soil rot is known (Barksdale
1974; Barksdale and Stoner 1981; Gardner 1988) but is generally polygenic and
difﬁcult to transfer into commercial cultivars. However, such forms of resistance (i.e.
horizontal) may be durable as opposed to resistance based on single genes (i.e.
vertical). If horizontal-type resistance can be successfully incorporated into commercial
lines, fungicide input can be further reduced. For example, O’Leary ( 1985) has shown
resistance to early blight in Dr. Gardner’s advanced lines is governed by the interaction
of infection efﬁciency, lesion area, latent period, and sporulation capacity. This rate-
reducing resistance can be combined with a reduced-sprays program to achieve control
(O’Leary 1985). Likewise, Barksdale and Stoner (1981) demonstrated resistance to
anthracnose provided control of fruit rot equivalent to 3 - 7 fungicide applications.

Biotechnological advancements also hold promise for enhanced disease control.
Anti-sense polygalacturonase expression in transformed tomato fruit limits fruit rot

(Kramer et al. 1992). Chitinase (Broglie et al. 1990) and other pathogenesis related

 

 

 

 

 

 

 

196

proteins can also be genetically engineered into tomato. Accumulation of Chitinase and
other anti-fungal proteins has been associated with resistance to Altemaria solani
(Lawrence and Tuzun 1992). This form of resistance is likely to be rate-reducing and
research would need to elucidate how engineered resistance can best be deployed so it
remains durable. Stewardship of non-renewable resources (i.e. effective genetic
resistance, natural or otherwise) may beneﬁt from an understanding of the population
diversity of the ﬁmgal pathogen. Likewise, resistance may remain durable if deployed
within integrated disease management systems. Research is required to assess the
diversity of A. solani, R solani, and important Colletotrichum species.

Biological control holds some promise but considerable basic research is
required to understand the dynamics of site speciﬁc deployment and mechanisms by
which introduced organisms provide control.

Advances in integrated disease management of bacterial disease of tomato may
also be accomplished with genetic resistance and biotechnological advances. To date,
acceptable levels of genetic resistance in tomato has not proven durable (Lawton and
MacNeill 1986, Wang et al. 1990). Considerable basic research is required to
understand the interaction between the bacteria and host. Identiﬁable resistance in the
host is often governed by a single dominant gene that speciﬁcally interacts with a
dominant gene for avirulence in the bacteria. However, avirulence genes may be
gratuitous (Gabriel 1989) and simply associated with genes for pathogenicity that
govern basic compatibility (Heath 1991). Loss of an avirulence gene in such cases
may be of no ecological importance to the bacteria other than circumventing a
hypersensitive host- defence response. Alternatively, genes that elicite a defence

response may be important as a component of virulence. The REP-, BOX-, and ERIC-

 

 

 

197 1

PCR appear to provide a survey of the chromosomal structure of bacteria. Speciﬁc
rep-PCR ﬁngerprint proﬁles may be associated with host speciﬁc virulence genes
(Waney et al. 1991) or species speciﬁc genes (Heath 1991) that function in a positive
manner to incite disease on speciﬁc hosts. A fundamental understanding of the basic
mechanism by which the bacteria cause disease will likely be arrived at by ﬁrst
elucidating the population structure of the pathogen. Once classiﬁed into ”lineages"
(Levy et al. 1991; Leach et al. 1992), experiments can be designed to elucidate the
mechanisms of basic compatibility and this knowledge may be useful to develop
resistance. Fundamental research is also required to determine how host resistance (i.e.
basic resistance sensu Heath) may impact basic compatibility of the pathogen.
Characterization of the population diversity of Cmm and Pst should also be
future research. Ability to describe variability of each pathogen may be of practical

use for the deployment of host resistance (Levy et al. 1991; Leach et al. 1992).

have the largest short-term impact. The REP-, BOX-, and ERIC-PCR can provide a
framework for elucidating important sub-populations that must be detected. Serological
or DNA hybridization based detection systems may then be devised. Diagnostic probes
speciﬁc for Pst (Cuppels et a1. 1990) and Cmm (Thompson et al. 1989) have been
developed but additional research is required to integrate their use.

Management of early blight, anthracnose, soil rot and bacterial diseases of
tomato is possible with current levels of technology and knowledge. However,
additional research is required to expand and integrate this technology and knowledge,

and formulate advanced integrated disease management strategies in tomato production

systems.

1
l
‘ Detection and identiﬁcation protocols also need to be advanced and will likely

 

 

198

LITERATURE CITED

Barksdale, TH. 1974. Evaluation of tomato fruit rot resistance to Rhizoctonia soil rot.
Plant Dis. Rep. 58:406-408.

Barksdale, T. H., A. K. Stoner. 1981. Levels of tomato anthracnose resistance
measured by reduction of fungicide use. Plant Dis. 65:71-72.

Broglie, R.D., et a1. 1990. Activation of a bean Chitinase promoter in transgenic
tobacco plants by phytopathogenic fungi. The Plant Cell 2:999-1007.

Cuppels, D.C., R.A. Moore, and V.L. Morris. 1990. Construction and use of a
nonradioactive DNA hybridization probe for detection of Pseudomonas syringae pv.
tomato on tomato plants. Appl. Env. Microbiol. 56:1743-1749.

Gabriel, D.W. 1989. The genetics of plant pathogen structure and host-parasite
speciﬁcity. Pages 343-379 in: Plant-Microbe Interaction III. T. Kosuge and E.W.
Nester, eds. Macmillan Publishing Co., New York

Gardner, R.G. 1988. NC EBR—l and NC EBR-2 early blight resistant tomato breeding
lines. HortScience 23:779-781.

Goode, M.J., and M. Sasser. 1980. Prevention- The key to controlling bacterial spot
and bacterial speck of tomato. Plant Dis. 64:831-834.

1 Heath, M.C. 1991. The role of gene-for-gene interactions in the determination of host
species speciﬁcity. Phytopathology 81:127-130.

Kramer et a1. 1992. Postharvest evaluation of transgenic tomatoes with reduced levels
of polygalacturonase: processing, ﬁrmness and disease resistance. Postharvest Biology
and Tech. 1:241—255.

Lawrence, CB, and S.Tuzun. 1992. Differential accumulation of chitinases, B-1,3,-
glucanases and osrnotins in tomato varieties resistant and susceptible to Altemaria
solani. Phytopathology 82:1101 (abstr.).

Lawton, M.B., and B.H. MacNeill. 1986. Occurrence of race 1 of Pseudomonas
syringae pv. tomato on ﬁeld tomato in southwestern Ontario. Can. J. Plant Pathol.
8:85-88.

Leach, J.B., M.L. Rhoads, CM. Vera Cruz, F.F. White, Mew, T.W. and Leung, H.
1992. Assesrnent of genetic diversity and population structure of Xanthomonas oryzae
pv. oryzae with a repetitive DNA element. Appl. Environ. Microbiol. 58:2188-2195.

Levy, M., J. Romao, M.A. Marchetti, and IE. Hamer. 1991. DNA ﬁngerprinting with
dispersed repeated sequences resolves pathotype diversity in the rice blast fungus. The
Plant Cell 3295-102.

 

 

 

199

O’Leary, DD. 1985. Effects of fungicides and host resistance on epidemics of tomato
early blight. Ph.D. Thesis. North Carolina State University. 175 pp.

Thompson, B.T., J .L. Leary, and W.C. Chun. 1989. Speciﬁc detection of Clavibacter
michiganense subsp. michiganense by a homologous DNA probe. Phytopathology
14:311-314.

Waney, V.R., M.T. Kingsley, and D.W. Gabriel. 1991. Xanthomonas campestn's pv.
translucens genes determining host-speciﬁc virulence and general virulence on cereals
identiﬁed by TnS-gusA insertion mutagenesis. Mol. Plant-Microbe Interact. 42623-627.

Wang, J.F., J.B. Jones, J.W. Scott, and RE. Stall. 1990. A new race of the tomato
group of strains of Xanthomonas campestris pv. vesicatoria. Phytopathology 80:1070
(abstr).

 

 

 

Table A1: Mean squares from analysis of variance for yield and fruit quality of fresh

 

APPENDIX A

market tomato (FMT) in 1992 using 4 replications.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Source FMT FRUIT QUALITY AND YIELD (MT/HA)
Varizliﬂity df LARGE %LRG MED #1 #2 CULL
#1 #1 #

Rep. 3 31 N8 280 NS 14 NS 1965 ' 11 NS
Rotation (R) 1 979 ' 474 NS 0.5 NS 85 NS 4 NS
Error 8 3 105 86 11 197 44
Tillage (T) l 916 NS 225 NS 31 ' 671 ' 20 NS
R x T 1 671 NS >1 NS 43 " 794 ' 278 “
Error b 6 231 NS 191 3 81 16 NS
Fung.(F) 6 110 ' 80 * 3 NS 137 * 17 NS
R x F 6 5 NS 26 NS 2 NS 12 NS 25 NS
T x F 6 61 NS 50 NS 4 NS 18 NS 6 NS
RxTxF 6 56 NS 32 NS 4N8 42 NS 14 NS
Error c 72 45 32 4 50 12

 

 

 

 

2.2... F—test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively.
N S, non-signiﬁcant.

" Fruit were sorted for large #1, % large #1, medium #1, #2 and culls.

200

 

 

 

201

 

TABLE A2: Effect of rotation and tillage on marketable fruit of fresh market tomato
in 1992 using 4 replications.

 

 

 

 

 

 

 

 

 

 

 

 

 

LARGE NO.1 MTIHa H
TILLAGE MT/H % MEDIUM No.2 CULL
a NO.1
NO ROTATION 19.6 29 8.5 22.5 17.2 1
WITH 25.5 33 8.7 24.3 17.6 \
ROTATION
PVALUE 0.055 0.10 <0.30 <0.30 <0.30 \
CT 19.7 29 8.1 20.9 17.0 l
ZT 25.4 ' 32 9.2 25.8 17.8 1
L P VALUE 0.09 <0.30 0.02y 0.032 W

 

 

 

 

’ interaction is signiﬁcant (R- x CT=8.65; R+ x CI‘=7.56; R— x ZT=8.47', R+ x ZT=9.84;
LSD=1.17 P=0.05).

2 interaction is signiﬁcant (R— x CT=22.7; R+ x CI‘=19.2; R— x ZT=22.3; R+ x ZT=29.4;
LSD=5.89 P=0.05).

TABLE A3. Effect of frmgicide treatment on marketable yield of fresh market tomato
in 1992 using 4 replications.

 

 

 

 

 

 

 

 

 

 

FUNGICIDE LARGE NO.1 MT/Ha 1
TREATMENT MT/H % MEDIUM No.2 \ CULL\
a NO.1
LWEEKLY 26.2 34.5 8.5 25.0 1 15.77
L DSV 25H 24.2 31.9 8.6 23.7 17.31
Q0 SPRAY 18.5 28.4 8.1 17.6 17.7 1
LP VALUE 0.03 0.03 <0.30 0.02 0.22 1
LLSD;P=.05 4.7 4.0 5.0 l \

 

 

 

 

 

 

 

202

TABLE A4: Mean squares from analysis of variance for yield and fruit quality of
processing market tomato (PRT) in 1992 using 4 replications.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Source PRT FRUIT QUALITY AND YIELD (MT/HA)‘

Van-2:111), df RIPE GREEN CULLS % RED
Rep. 3 156 NS 3394 * 13 NS 1980 NS
Rotation (R) l 36 NS 34 NS 0.4 NS 505 NS
Error 8 3 140 192 12 590
Tillage (T) l 773 ... 1564 " 30 * 580 NS
R x T l 722 407 NS 22 ' 1054 '
Error b 6 16 124 3 124
Fungicide (F) 6 41 NS 635*“ 31 .... 394 ...
RxF 6 6N8 43 NS 2N8 49NS
T x F 6 16 NS 18 NS 12 NS 55 NS
RxTxF l6 17 NS l 104 NS 16NS 30NS
Error c l 72 l 23 l 79 l 3 l 85

 

 

F—test signiﬁcant at P = 0.05, P = 0.01 or P = 0.001, respectively-
NS, non-signiﬁcant.

" Fruit were sorted for color (ripe or green) and quality (marketable fruit or culls).

 

 

e.

 

203

TABLE A5: Effect Of tillage on marketable yield of processing tomato cv. Heinz 8780
in 1992 using 4 replications.

 

 

 

 

 

 

 

 

 

MT/Ha

TILLAGE %

RIPE GREE CULLS RED
N
NO ROTATION 12.1 24.1 2.9 32
WITH 10.9 25.2 2.7 28
ROTATION

P VALUE <0.30 <0.30 <0.30 <0.30

CT 8.9 20.9 2.3 27

ZT 14.1 28.4 3.3 32

P VALUE 0.0004y 0.01 <O.02z 0.07

 

 

 

 

 

 

3' interaction is signiﬁcant (R— x CT=l2.0; R+ x CI‘=5.8; R- x ZT=12.1; R+ x ZT=16.1;
LSD=2.54 P=0.05).

‘ interaction is signiﬁcant (R- x CT=2.8', R+ x CI‘=l.8', R— x ZT=2.9; R+ x ZT=3.7;
LSD=0.98 P=0.05).

TABLE A6. Effect of fungicide treatment on marketable yield of
processing tomato in 1992 using 4 replications.

 

 

 

 

 

 

 

FUNGICIDE MTlHa
TREATMEN % RED
rRIPE \ GREEN 1 CULLS 1
\ WEEKLY \ 9.7 \ 33.5 1 1.1 1 22.0
usv 2er 1 9 7 I 28.2 \ 1.3 \ 24.0

\ NO SPRAY \ 108 1 13.3 1 4.9 \ 35.8

VP VALUE \ 012 \ >0.001 I >0.001y \ >0.001
Imp-rams \ \ 6.3 \ 1.3 \ 6.5

 

 

V The fungicide x tillage and fungicide x tillage x rotation interactions were signiﬁcant.

 

 

 

APPENDIX B

EFFECT OF TILLAGE AND FUNGICIDE ON THE INCIDENCE
OF BACTERIAL SPECK

Fresh market tomato fruit were harvested weekly in 1992 and fruit with
bacterial speck symptoms was weighed (Chapter 2). Preliminary analysis of data
indicate weekly applications of fungicide resulted in elevated levels of speck, caused
by Pseudomonas syringae pv. tomato.

Zone tillage in non-rotation plots reduced speck incidence. Thus, cultural
practices also have some inﬂuence on the populations of bacteria able to infect tomato.

204

 

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APPENDIX B CONTINUED

EFFECT OF TILLAGE ON THE EARLY INFESTATION OF PLOTS BY THE
COLORADO POTATO BETTLE

Total incidence of Colorado potato beetle was monitored early in the 1991 and
1992 seasons. In 1992, rotation alone did not seem to impact the number of fresh
market tomato plants with CPB but reduced the. incidence on processing plants. In
contrast, zone tillage dramatically reduced CPB counts on all tomato plants. Excessive
rye residues on the soil surface may disrupt the CPB from locating its host.

206

 

 

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207

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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APPENDIX C

DIRECT ANALYSIS OF BACTERIA ON MEDIA AND IN PLANT LESIONS

PURPOSE: To determine if ﬁngerprint patterns can be generated using whole
cells collected from media and from lesions on plant tissue to identify unknown
isolates.

MATERIALS AND METHODS: Clavibacter michiganensis subsp.
michiganensis (CMM 936), Xanthomonas campestris pv. vesicatoria (Xcv 982, 736),
Pseudomonas syringae pv. tomato (PST 915), and unknown cultures were initiated on
King's B (KB), nutrient yeast dextrose agar (NYDA), SCMZ-t (selective for CMM), or
Tween B (selective for XCV). After 7 days of growth, a low number of cells were
collected and placed directly into epindorph tubes containing reagents for the
polymerase chain reaction and primers corresponding to BOXA. Samples were subject
to conditions outlined in Chapter 3.

Samples of tomato from a commercial ﬁeld with a presumptive bacterial
epidemic was brought to the lab. Lesions were cut from fruit and a DNA miniprep
was performed. One 111 of extract was subject to PCR as described above.

DNA extracted from isolates proven to be true pathogens by Kochs postulates
were used as positive controls.

RESULTS: CMM, XCV and PST cells collected from media yielded DNA
ﬁngerprints identical or similar to control DNA when subject to BOX-PCR. XCV
collected from NYDA did not yield an acceptable pattern possibly due to high levels
of polysaccharides on this rich medium. Extract from unknown lesions generated
fingerprint patterns similar to XCV control DNA. Unknown sarnples isolated from an
unknown spot on tomato fruit and collected from single colonies on NYDA generated

unique ﬁngerprint patterns as compared CMM, XCV and PST and could not be
identiﬁed.

DISCUSSION and CONCLUSIONS: Cells of bacteria on selective or non-
selective media can be rapidly identiﬁed using BOX-PCR. Our experience has been
that colonies of CMM, XCV and PST when isolated from symptomatic plants appear
on selective or non—selective media within 4, 2 to 4, and 2 days, respectively. Taken
together, these data suggest rapid non-presumptive diagnosis of bacterial colonies can
be accomplished for Gram positive and Gram negative phytopathogenic bacteria if
control patterns are available for comparative analysis. Identiﬁcation of bacteria
directly from plant lesions is also possible but contaminating bacteria appear to alter
patterns such that the ﬁngerprint proﬁles are not identical to control DNA. This
technique holds considerable possibilities for routine and rapid identiﬁcation of
unknown isolates.

I thank Nancy Fichter for conducting this and other similar experiments.

208

 

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APPENDIX D

SPECIFIC GENOMIC FINGERPRINTS OF PHYTOPATHOGENIC
XANTHOMONAS AND PSEUDOMONAS PATHOVARS AND STRAINS
GENERATED WITH REPETITIVE SEQUENCES AND PCR.

210

 

211

AI’PLIII) \.\'1) E.\'\1R0.\'MI:.\'TAI M1(ROIII()1()(I\ July 1994. p. 2286—2295 Vol. OI). No. 7
(1119‘) 224(ll94/$fl4. 1111+“
Cop\right-L C 1994. American Socicu for Microbiology

Speciﬁc Genomic Fingerprints of Phytopathogenic Xanthomonas
and Pseudomonas Pathovars and Strains Generated with
Repetitive Sequences and PCR

FRANKJ. LOUWS. '* DENNIS W FULBRIGHT. "' ‘ CHRISTINE TAYLOR STEPHENS '
AND FRANS J. DF. BRUIJN

Dcpamnenl afo 8011111) and Plan! Pal/lolog).' Michigan Slate Ullil‘t’l‘an D-epanmenl of Enug) Plan! Research
ralon.J Depnmncnl oj Microbiologi. (Ind Nalional science Foundalian C cnlel for
oMicrobia/ Ecology ‘ Michigan Stale Unitersitix Earl Lansing. Michigan 48824

Received 22 December 1993/Accepted 12 April 199-t

DNA primers corresponding to conserved motifs in bacterial repetitive (REP. ERIC. and BOX) elements and
PCR were used to show that REP-. ERIC-, and BOX-like DNA sequences are widely distributed in
phytopathogenic Xanthomonas and Pseudomonas strains. REP-. ERIC-. and BOX-PCR (collectively known as
rep-PCR) were used to generate genomic ﬁngerprints of a variety of Xanlhamonas and Pseudomonas isolates and
to identify pathovars and strains that were previously not distinguishable by other classiﬁcation methods.
Analogous rep-PCR-derived genomic ﬁngerprints were generated from puriﬁed genomic DNA. colonies on agar
plates. liquid cultures. and directly from lesions on infected plants. REP-. ERIC-. and BOX-PCR-generated
ﬁngerprints of speciﬁc Xanthomonas and Pseudomonas strains were found to yield similar conclusions with
regard to the identity of' and relationship between these strains. This suggests that the distribution of REP-.
ERIC-. and BOX-like sequences in these strains is a reﬂection of their genomic structure. Thus. the rep-PCR
technique appears to be a rapid. simple, and reproducible method to identify and classify Xantliamonas and
Pseudomonas strains, and it may be a useful diagnostic tool for these important plant pathogens.

 

Plant pathologists are faced with the important challenge to sequence (24. 49). and the recently discovered 154-17p BOX
discern plant- -pathogenic variants within the species Xun- element (32. 41). REP. ERIC. and BOX elements have the
diamonds camperlris (Pammel 1895) Dowson 1939. Xanlhrmio- potential to form stem-loop structures and may play an impor»
nas oryzae ex lshiya ma 1922. and Pseudomonas syringae van tant role in the organization of the bacterial genome (33. 34.
Hall 1902. These species are currently subdivided at the 40).Gcnome organization is thought to be shaped by selection.
infraspeciﬁc level into 143. and 45 pathovars. respectively and thus the dispersion of the REP. ERIC. and BOX~ sc-
(II 52 69. 70). Pathovars2 within each species cannot be quences may beindicative ofthe structure and evolution olthc
reliably distinguished by their cellular metabolism or othcr bacterial genome (19. 33. 34. 40). On the basis of this assump-
phenotypic characteristics (10. 43. 54. 55). Therefore. they are tion and knowledge about the clonal nature and population
classiﬁed on the basis of their distinctive pathogenicity to one dynamics of pathogenic bacteria (1. S. if). 37. 38. 48). we
or more host plants (69). Unfortunately. identiﬁcation based hypothesized that if each evolutionary specuilized line. or
on pathogenicity tests can be inconclusive and open to alter- pathovar. of the pathogen had a unique distribution or ar-
native interpretations (15. 17. 37). Several attempts have been rangcment of repetitive sequences.throughout the genome. we
made to classify pathovars and strains by using alternative should be able to generate genomic fingerprints that correlate
features of the pathogen. Serologic testing (3. 4). fatty acid with a speciﬁc lineage or pathovar. . .
proﬁling (51 59). genomic and plasmid DNA analysis(5 8. 21. In this paper.-we demonstrate the utility of the PCR

23 31. 36—38 44). and protein analysis (55 56. 58) have been technique with primers corresponding to ubiquitous repetitive
used to classify pathovars and strains of different species. DNA sequences (rcp- PCR [7. 32. 63]) to generate schIlIc
However. these techniques are often time-consuming. too DNA ﬁngerprints of Xunl/Iommmt and Pseudomonas patho-
expensive, or too insensitive for use in routine diagnosis. vars and strains. In addition. we showthe potentialol rcp- PCR
Therefore. we have been interested in developing new mcth- fingerprinting as a diagnostic tool and In determining whether
ods to rapidly identify and classify closely related pathogenic pathovars represent a Single evolutionary line or are composed
bacteria on the basis of genomic ﬁngerprinting approaches. of several lInes that have converged to a similar pathogenic

Families of repetitive DNA sequences are dispersed phenotype.
throughout the genome of diverse bacterial species (32. 4(1. ()2).
Three families. unrelated at the DNA sequence level. have ‘
been studied in more detail. namely the 35- to 410-bp repetitive MATERIALS AND MEI "ODS
egtragcnic palindromic (REP? sequence (18‘ 23' the ERICO Bacterial isolates. Sources of and relevant information on
1-7-hp enterobacterial repetitive Intergcnic conscnsus( ) bacterial isolates or genomic DNA used in this study are listed

in Table 1. All bacteria were stored at —7(l°C in glycerol and
streaked on nutrient-yeast- -dextrosc agar (X. (HI/Hpt'ffllt and A.

. corresponding author. Present address: Center For Microbial ’ . 3” We focused on
ECOIOgy/MSU- Plant Research Laboraton. Rm 3110 Plant Biology oryzut [27]) or Kings Bilgar (P “”"SI‘I‘ i i) d (11
Bld e. Michigin State University EN L‘InginL Ml 48824 phone. pathovars that have been systematical y compare ani igvzga
(5|?) 353 2009. Fax: (517) 353.9168 Electronic mail address: sirgiilarphcnotvpc genotype orhoslmnth3 4.31 ..........

22473MGRGI msu.edu.

2286

 

212

 

 

|
V01. hf). l9)-l ,\.-f.\'TH().\I().\'.-lS AND I’SEL'I)().\I()\.A1.\ rep-PCR FINGERPRINTS 2287
TABLE 1. XII/Illrrmiunat and I’semlmnunm isolates or DNA used in this stud\
SPL'CIL‘\ and
pulhmar Isolate" Hust Location \ r isolated Sourcc' Rctcrcitcc
X. cunIpesII-n
c ATCC 331511-1’ ‘ Pm: InI'III/Ii ‘ "
raminis ATCC 29091“ ' Durn'ln glrnnI'I-Inu :TCE i:
translucens ATCC ltl77ll' Bar c\ ATC( -
umpesiris ATCC 33913" ' BrtIxtica ('IInI/ict'lnt UK ATCC 111
campestris NI (BZ—l)’ Bmwcu sp OR JH “(1
CdeCSIfh Xec 2D52tl Wild mustard CA JT 7”
campestris Cabbacu M] [00) - ’ ‘
campestris ch X98 Cabbage M1 199(1 JT iiii: :iaiii
phaseoli ATCC 9563‘ ' (NCPPB 3035) Bean ATCC iii ' i '
pﬂascop X35: (NCPPB 206-t) can JH 311
p aseo I 'cp I405 Bean ‘ " — '
begoniae X33 (118361.67. JM) Begonia PAL] i333 if: It?“ stud)
pelargonii X-l Gera Iu KS 1986 RD 9
pelargonii 5-2-4 Geranium Israel 1987 MD 9
pclargonii X—S Geranium M1 1986 RD 9
pclurgonii _'-1-7 Geranium NY 1957 MD 9
pelargonii 6945-s Geraniu FL JBJ
pelargonii P854391) Gemnru FL 1985 JM
."1 xcrif Citr JH 211
curt B . XCN-F Citrus JH 21)
vesicatoria Xt-3l Pepper OK 1990 CB
vesicatoria Xv9l Pepper Taiwan JBJ
vesicatoria Xcv lh' Pepper OH 1993 SM
vesicatoria Xt 92-16 Pepper FL RS
vesicatoria Sp oh Pepper seed GA 1993 GO
vesicatoria X 855 omato MX JBJ
Icaioria ATCC 11633 Pepper NJ 1047 ATCC
vesica oria ATCC 35937" Tomato N.Z. 1955 ATCC 1(1
chIcatorIa ATCC 11551 Tomato IN 1943 ATCC
X. oryzae
oryzae ATCC 43837" Rice ATCC 53
oryztcola ATCC 4907? Rice ATCC 53
P. syringae
morsprunorum Pin 7 Cherry MI 199' N
morsprunorum m 36 Cherrv M1 1991 AJ
morsp no Pm 567 Cherry UK M
syringae Pss ll Cherrv M1 199' A]
syringae P55 11 Cherry M1 1991 AJ
svringae P55 66 Cherry MI ‘99] M
syringae ss 1 Cherry MI 1991 A]
tomato Psi 82-14 UGA Tomato GA 1933 RC‘
tomato Pst 88-37 UGA Tomato GA 1988 R0
tomato Pst 88-40 UGA Toma GA 1938 R0
tomato Pst 856 Tomato MI 1992 This study
tomato Pst 915 TOITINO MI 1993 This “my

 

" ". type strain for the species: 9. pathovar reference strain: ‘. received as DNA from J. Smith. Michi

Department of Agriculture. Beltsville Md.: ".
_ " ATCC. American Type
Isolated fro Phasealur i-ulgan‘s seed): KD. K D
Gulf Coast Research Center. University of Florida: JM. J. Miller. Florida
State University: SM. 5, Miller. Ohio State University: RS. R.
State University. RG. R. Gitaitis. University of Georgia.

Preparation of DNA. Total genomic DNA was prepared by
using a modiﬁcation of the procedure of Ausubel et al. (2).
Brieﬂy. cultures were grown in 40 ml of Luria-Bertani medium
(46) for 24 to 48 h at 27°C. Cells were lysed in sarcosyl buffer,
and the resulting lysate was treated with pronase. DNA was
puriﬁed with a solution of CTAB—NaCI (10% cetyltrimethyl-
ammonium bromide in 1 M NaCl) followed by chloroform and
phenol-chloroform extractions and was recovered by isopropa—
nol precipitation. redissolved in TE (10 mM Tris. 1 mM EDTA
[pH 8.0]). and quantiﬁed spectrophotometrically.

Culture Collection: JH. J. Hartung (see footnote 11): JT. .1. T up (see footnote a) LA
unbar. Michigan State University: M
‘ Depanment of Agriculture and Consumer Services. Gar
Stall. University of Florida: 00.

gan State University: I. received as DNA from J. Hartung. U.S.
solute codes in parentheses are known alternative codes.

Manador. Michigan State University (pathovar

search 0 . J. Jones.

a

. nder. Oklahom
G. O'Kecfe. Georgia Department of Agriculture; M. A. Jones. Michigan

Ampliﬁcation and separation of DNA bands. Primer se-
quences corresponding to REP (REPlR-I [5’-1111CGICGI
CATClGGC-B’] and REPZ—I [5’-1CGlC1'TATCIGGCCfAC-
3']) were provided by J. R. Lupski (63) or synthesized as
described below. Primer sequences corresponding to ERIC
(ERICIR [5’-ATGTAAGC1‘CCTGGGGATTCAC-3'] and
ERICZ [5’-AAGTAAGTGACTGGGGTGAGCG-3‘]) were
synthesized with a DNA synthesizer (model 3808; Applied
Biosystems. Foster City. Calif.) by the Macromolecular Struc-
ture. Sequence and Synthesis Facility at Michigan State Uni-

 

213

3355 LOUWS ET AL.

versity. The primer sequence corresponding to BOXA. a
subunit of the BOX element (41) (BOXAlR [S’-CTAC
GQCAAGGCGACGCTGACG-S'J). was also synthesized at
Michigan State University or provided by .l. R. Lupski (32)
PCR conditions were as previously described (7). The PCR
protocols with REP. ERIC. and BOX primers are referred to
as REP-PCR. ERIC-PCR. and BOX-PCR. respectively and
rep-PCR collectively. PCR ampliﬁcation was performed with a
model 1 llls Tempcycler ll (Coy Corp.. Grass Lake. Mich.) by
using the following cycles: 1 initial cycle at 95°C for 7 min: 30
cycles .or dcnaturation at 94”C for l min. annealing at 44. 52. or
{3‘1' lot 1 min with REP. ERIC. and BOX primers. respec-
ll\’L‘l_\’..1ll‘lLl extension at 65°C for 8 min with a single ﬁnal
extension cycle at 65°C for 15 min and a ﬁnal soak at 43C. PCR
mixtures were overlaid with 25 pl of mineral oil (M3516:
Sigma).

A 5- to S-pl portion of ampliﬁed PCR product was separated
hy gel electrophoresis at 4°C on 1.5% agarose gels in 0.75X
[AL-butler (46) for to n at 5 V/cm. stained with ethidium
bromide. and photographed on a UV transilluminator with
Polaroid type 55 ﬁlm. Fingerprints generated from diﬁercnt
strains were compared visually.

Reproducibility of DNA ﬁngerprints. Fingerprint proﬁles
generated from independent DNA preparations extracted
lroin single-colony cultures at different times (over a period of
severatnmnths) were run side by side on an agarose gel to
determine their reproducibility. A protocol involving the direct
assay of whole cells exuding from plant lesions. suspended in
water. or collected directly from solid media was also tested.
For this protocol. tomato or geranium leaves with apparent
symptoms of bacterial speck or bacterial wilt. respectively.
were surface sterilized for l min in 0.53% sodium hypochlorite
and rinsed three times in sterile distilled water. Lesions were
dissected and macerated in 300 pl of water and allowed to
stand for 15 min. Then i p.l of the resultant suspension was
added to the PCR mixture. DNA from known pathogenic
isolates of I), .nxn‘ugae pv. tomato and X. campestris pv. pelar-
gonn was used as a positive control and run nut to the
unknown samples on agarose gels for comparison. For the
geranium sample. the resultant suspension was also streaked
on nutricnl—yeast-dcxtrose agar and cells from yellow colonies
were directly assayed as described below. The REP-PCR
proﬁle generated from DNA of an isolate of X. campestris pv.
vesicatoria (P-US) was compared with the proﬁle generated
trom the same isolate submitted to our laboratory as a
Suspension in sterile water. A l-|J.l sample of the suspension
was added to the PCR mixture. Likewise. samples of cultures
suspected to be X. cum/wx/rix pv. pelurgonii were submitted as
cultures on solid media. Bacterial cells were collected directly
from a colony by using a l-al disposable inoculating loop and
rcsuspended in the PCR mixture. Fingerprints were resolved
on an agarose gel within 24 h of receipt of the sample. DNA of
a known X. campesrris pv. pelargonii isolate was used for
CCIl‘liplll‘lSOH.

RESULTS

rep-PCR DNA ﬁngerprints clearly distinguish dilferent
pathovars. Primers corresponding to conserved DNA se-
quences of REP elements. BOXA subunits of BOX elements.
and ERIC sequences annealed to genomic DNA and gener-
ated unique genomic ﬁngerprints for each pathovar
matin- and .\'. tutu/Mains tested. The ﬁngerprint patterns of
representative strains (Table l) ofX. onene pv. oryzae and pv.
oryzicola and .\‘. t‘tllll[)('S/IT3 pv. poae. pv. graminis. pv. translu-
cens. pv. campestris. pv. phaseoli. pv. citri. pv. begoniae. pv.

Al’PL. ENVIRON. MICROBIOL.

4072
3054 -

2036- y,
1636. j_

101B-

’
-

   

HO. 1. rep-PCR ﬁngerprinting patterns from genomic DNA of X.
uricuc and .\'. cum/Jesuit isolates. Tic REP-PCR. BOX-PCR, and
ERlC-PCR patterns are shown in panels A. B. and C. respectively. A
5- to S—pl portion ol’ each of the rep-PCR mixtures was loaded onto a
1.5% agarose gel. The resulting electrophoretic patterns of isolates X.
ul]‘:tIL' pv. oryzae ATCC 43837 (lanes 1) and pv. oryzicola ATCC 49072
(lanes 3) and X. campestris pv. poae ATCC 33804 (lanes 3): pv
graminis A'l'CC 3909| (lanes 4); pv. translucens ATCC 10770 (lattes
5): pv. campestris ATCC 33913 (lanes (3). X6 (lanes 7). ZDSZU (lanes
8). JTl (lanes 9). and S98 (lanes Ill): pv. phuseoli ATCC 9563 (lanes
11). X35 (lanes 11). and 805 (lanes l3): and pv. citri A chZ (lanes 14)
and B Xc 34 (lanes 15) are shown. The control lane. not labeled.
represents the same rep-PCR but laclu'ng template DNA. The left and
right lanes labeled 5 show the DNA molecular size marker (I-kh
ladder: Gibco-BRL): the sizes are indicated in base pairs. Arrowheads
identify similarities or dilferences among selected isolates as outlined
in the text.

pelargonii. and pv. vesicatoria are shown in Fig. I and 2. The
REP-. BOX-. and ERIC-PCR yielded 5 to more than 20
distinct PCR products. ranging in size from approximately 100
' kb. Dilferences among pathovars were assessed

to over a
visually on the basis of the migration patterns of PCR products.

 

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FIG. 2. rep-PCR ﬁngerprinting patterns from genomic DNA of X.
mmpcxlrrlr isolates. The REP-PCR. BOX-PCR. and ERlC~PCR pat»
terns are shown in paneLs A. B, and C. respectively. A 5- to S—pl portion
of each of the rep-PCR mixtures was loaded onto a 1.5% agarose gel.
The resulting clectrophoretic patterns of isolates pv. begoniac X3
(lanes l); pv. pelargonii X-l (lanes 2). 5-2—‘1 (lanes 3). X-S (lanes 4).
5-l~7 (lanes 5). 6945~S (lanes 6). and 1385—390 (lanes 7); and pv.
vesicatoria isolate Xv3l (lanes 8). Xv9l (lanes 9). XCVIS (lanes [0). Xv
93-16 (lanes ll). Sp 66 (lanes [2). Xv SSS (lanes 13), ATCC 11633
(lanes 14). ATCC 35937 (lanes l5). and ATCC llSSl (lanes 16) are
shown. Other details are described in the legend to Fig. I.

X. myzae pv. oryzae and pv. oryzicola constitute a single
species on the basis of DNA-DNA hybridization studies (52)
and share many phenotypic features (60) but incite different
symptoms on rice plants (42). REP-. BOX-. and ERIC-PCR
clearly diﬂerentiated the pathovar reference strains of X.
oryzae pv. oryzae and pv. oryzicola (Fig. 1. lanes l versus lanes
2). Fingerprint proﬁles generated with each primer set were
complex and very different between the two pathovars. In total.
over 60 distinct bands were visualized. and only one major
PCR product. generated by BOX—PCR and highlighted by an
arrowhead (Fig. lB. lane l). appeared to comigrate in both

.\'.lNTHO/\ION.lS AND PSEUDOAIONAS rep—PCR FINGERPRINTS 223‘)

strains. Therefore. no obvious relationship between the two
pathovars could be surmised on the basis of the REP-. BOX-.
or ERIC-PCR ﬁngerprint patterns.

X. coin/mitts pv. poae. pv. graminis. and pv. translucens are
closely related on the basis of DNA hybridization and pheno-
typic studies (53. 59) but are classiﬁed as distinct pathovars
primarily on the basis of their host range (I 1. l2. ()9). In our
study. the pathovar reference strain of X campestris pv. poae
yielded distinct ﬁngerprint proﬁles from those of the reference
strain. X. campestris pv. graminis (Fig. 1. lanes 3 and 4.
respectively). but the presence of several comigrating hands
suggested that X campestris pv. poae and pv. graminis are
closely related. The REP-PCR proﬁles of X. campestris pv.
graminis were comparatively simple. and at least four PCR
products. highlighted by arrowheads (Fig. 1A. lane 4). comi-
grated with bands generated from DNA of X. runrpesn-ix pv.
poae. BOX-PCR also yielded multiple bands of parallel mo-
bility (Fig. lB, lanes 3 and 4). Comigrating bands generated by
ERIC-PCR and highlighted by the arrowheads (Fig. 1C. lane
4) were also visible. but the overall pattcms were distinct. The

B X—. and ERIC-PCR proﬁles of X. campestris pv.
translucens (Fig. l, lanes 5) were found to be ditl'erent from
those of both X. campestris pv. poae and pv. graminis.

A third example highlighting the ability to detect distinct
xanthomonad pathovars is shown in Fig. 2. X. campestris pv.
begoniae and pv. pelargonii are distinct groups on the basis of
serologic (4). host range. phenotypic. protein electrophoretic.
and DNA hybridization (58) features. The REP-. BOX-. and
HRlC—PCRs (Fig. 2A, B. and C. respectively) generated com-
plex banding patterns from DNA of representative strains ofX.
campestris pv. begoniae (lanes l) and pv. pelargonii (lanes 2 to
7). and each primer generated very diﬁ‘erent patterns for the
two pathovars.

Pairwise comparisons of strains representative of P. tyn'ngar:
pv. morsprunorum. pv. syringae. and pv. tomato demonstrated
that the REP—. ERIC. and BOX-PCR ﬁngerprint proﬁles were
also distinct for different pathovars of P. syringae. P. syringae
pv. morsprunorum and pv. syringae incite the same disease of
stone fruits and. when isolated. cannot be differentiated except
by dilatory biochemical tests (35) and more recently with a
DNA probe (433). Our ﬁngerprint proﬁles readily distin-
guished between the two pathovars (Pic. 3. lanes 1 to 3 versus
lanes 4 to 7). Likewise. l’. stringae pv. syringae. an economically
insigniﬁcant pathogen oftomato, and P. mingae pv. tomato. an
economically important pathogen of tomato. can he distin-
guished by a speciﬁc DNA probe (7a) and rare—cutting restric-
tion enzymes (6a) but cannot be rapidly diﬁ‘erentiated by other
experimental means (26, 27). The REP-. BOX-. and ERIC-
PCR ﬁngerprint proﬁles clearly distinguished strains of P.
{ll/ingot: pv. syringae and pv. tomato (Fig. 3, lanes 4 to 7 versus
lanes 8 to 12).

Pairwise comparisons of any two pathovars tested demon-
strated that strains representative of a particular pathovar yield
DNA product patterns that are complex and easily distinguish-
able from patterns generated from strains belonging to any
other pathovar within the species X. oryzae. X. campestris. and
P. .wringae. Each set of primers (REP. BOX. and ERIC) was
effective in distinguishing different pathovars.

Intrapathovar variation. In contrast to the diversity of
ﬁngerprints observed among strains representative of different
pathovars. each set of primers yielded common banding pat-
terns among isolates witbin a pathovar. To determine the
intrapathovar diversity of DNA ﬁngerprints. we examined
several isolates of selected pathovars obtained from geograph-
ically distinct locations or isolated from the same geographic
area at dilfcrent times (Table l). -

 

 

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IG. 3. rep- PL R lingLIprintimg1 pallerns 11rorn genomic DNA 01 P
.wringm' isolates. The REP X- PCR. and l:‘ RlC- PCR patterns
are 51 ow II in panels A. B. and C. respectively. A5- to Seal portion of
each of the rep-PCR mixtures was loaded onto a 1.5'7bagarose gel. The
resulting electrophoretit‘ patterns of isolates pv. morsprunorum P111 7
(lanes 1). Pm 36 (lanes 2). and I’m 567 (lanes 3): pv. syringae Pss ll
(lanes >1), Pss ll (lanes 5). Pss (16 (lanes (I). and Pss 19 (lanes 7): and pv.
tomato Ps1 $114 (lanes 8). Pst 88-37 (lanes 1)). Pst 8.9-40 (lanes 10). Pst
SStI (lanes 11). and Pst 915 (lanes 12) :IIL shown. Latin 13 shows the
control Icattion laelking template DNA Other de1ails arL described 111
1hr: legend 11) Fig

The intrapathovar diversity of the isolates tested could be
grouped into two broad categories: (i) pathovars from which
isolates had nearly identical REP. BOX. and ERIC ﬁnger—
prints or from which isolates had overall unique proﬁles but
shared multiple bands 111' apparent equal mobility. and (ii)
pathovars from which isolates could be divided into groups
(i.e.. evolutionary lines) 111211 did not share common REP-.
BOX-. or ERIC-PCR handing patterns.

 

Al‘l’l.. ENVIRON. MICROBIOL.

Most of thc pathovars were found to belong to the ﬁrst
calcgory. For example. no notable diﬁcrcnccs were UbSCl'VCd
between the three isolates of X. campestris pv. phaseoli (Fig. 1.
lanes 1 l to 13) or between the thrLL isolates of P syringae pv
morsprunqum tested (Fig.3 .lanes 1 to 3). The analysis of si.\
isolates 0 Y. calripexn‘it pv.pela1gonii by the BOX- PCR
yielde idLnliL‘al proﬁles (Fig. 2B. lanes- 7 to 7). The REP- PCR
viL-ldL-d LI single additional band for isolates S- 2- 4. --1 7 and
PSS-S‘JO compared with the other three isolates Iesled (Fig.
2A. lanes 2 to 7). The ERIC-PCR of isolates 5-2-4 (Fig. 2C,
lane 3) and P85-390 (Fig. 2C. lane 7) yielded a single polymor-
phism (arrowheads) in contrast to the other isolates. l-or the
ﬁve isolates 01 P. mingac pv. toman tested (Fig.3
12). two patterns were apparent. lsolate 915 demlonstrated
some polymomhisms (accentuated by arrowheads in Fig. 3.
lanes 12) in contrast to the other four isolates anmined. but
the maonity of bands were analogous. Two distinct gioups of
I’. .9 I’ll! cpv. tomato strains have also been identiﬁed by
restriction fragment angth polymOIplIism analysis (8) and ﬁeld
inversion gel electrophoresis of DNA digested with rare-
L‘utting enzymes (6a).

Each X. t‘umpes/ris pv. campestris and pv. citri isolate tested
had unique RFZPL BOX-. and ERlC~PCR ﬁngerprint proﬁles.
but the presence of multiple bands of apparent equal mobility
suggested that isolates within each pathovar had a common
evolutionary heritage. For example. the ﬁve isolates of X.
caIIIpcsII-is pv. campestris (Fig. 1. lanes (1 to l0). including the
type strain for the pathovar and species (ATCC3 3913 [lanes
(1]). )shared multiple comigrating bands (aL‘L‘Lntuated by oppos-
ing arrows in lanes (1 and 10) but were highly diverse 101 the
remainder of the bands generatLd. Isolate X6. (lanes 7) was
highly similar to .lT1 (lanes 9), and both were similar to the

C type strain (lanes 6). Likewise. theX. camper/r15 pv. citri
pathotype A isolate (Xc62; lanes 14) and the pathotype B
isolate (Xc84: lanes 15) shared PCR products of equal mobility
(accentuated by arrowheads in lanes 15). but the overall
patteins weIe quite distinct (compare lanes 14 andl 5.)

The 10111 isolates of)” syringae pv syring ac (Fig. 3. lanes 4 to
7) had a number 01' REP-. BOX-. and ERJC- PCR products In
common. accentuated by the arrowheads in lanes 4. but
multiple additional bands were generated to yield strain-
speciﬁc proﬁles. Isolate Pss 11 (lanes 4) and P55 11 (lanes 5)
yielded apparently identical proﬁles with each type of primer
set. isolate Pss 66 (lanes 6) was highly similar to P55 1 l and P55
11 but yielded a number 01‘ unique PCR products. The
BOX-PCR (Fig. SB) proﬁles appeared more similar among
these three isolates than did the REILPCR (Fig. 3A) and
ERIC-PCR (Fig. 3C) proﬁles. In contrast. the REP». BOX-.
and ERlC-PCR patterns generated from DNA of P55 19 (lanes
7) were very diEcrent from each of the other three isolates.
although common bands (accentuated by the arrowheads in
lanes 4) of equal mobility could be identiﬁed.

The REP-. BOX-. and ERIC-PCR protocols provided sim-
ilar conclusions about the apparent relatedness among isolates
that yielded similar ﬁngerprint proﬁles. When isolates within a
pathovar demonstrated polymorphisms. each primer set of-
fered unique information. genLra1ing slrain- speciﬁc proﬁles.

The second categoiy of diversity was observed within the
pathovar X unnpemix pv. vesicatoria. The REP-. BOX—. and
ERI C-P CR protocols demonstIated that this pathovar is com—
posed ol at least two distinct gioups (Fig. _, lanes 8 to 16).
Within the ﬁIst group (group A [lanes 8 to 14]). isolates
obtained over time and from distant geographic sources in-
cluding Taiwan (Xc 91 [lanes 9]). Mexico (Xv SSS [lanes 14]).
and several states 01' the United States including Oklahoma
(Xv 3‘) [lanes 8]). Ohio (Xcv 18 [lanes 10]), Florida (Xv 92-16

 

 

 

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FlG <l ERIC- PCR ﬁngerprint patterns of X. “1111/11 1111': pt.
campestris 11nd pr. pelatgonii 11nd P, _\'11111L't11' pv.syr1ngae isolates
venerated lrom dilIL'tL'nt DNA preparations from single- colon_\ Lul-
ture. of the samL strr11in.Tl1e tesulting electropltoretic pattetn: ol
isolates .\. 1'11111pt'xlt11' pv. campestris .ITI (lanes 1 11111.1 2) 11111] $98 (lanes
3 and 1: .\i L'ilmpt'xlris pv. pelargonii X-l (lanes 5 11nd (t) attd 5 3»
(lanes 7 and S): attd P, 11'1‘1'113111'pv.syringae Pss ll (lattes U and 10). Pss
ll (lanes 11 and 13). Pss ()6 (lanes 13 11nd ll). .tnd Pss 19(l1tnes 15 and
1111 are shown. Other details are described in the legend to Fig 1

[lanes 11]). Georgia (Sp ()0 ll anes 12]) and an ATCC strain
(ATCC lit» 1 3 llanes 14]) were very similar to each other. with
vety few unique h11nds.No major diﬂerettces were noted
among these sttains when tlte REP- PCR protocol was used
(Fig. 3A). but the presence or absence of one or more unique
bands was noted when tlte BOX-PCR (I'ig. IR) and ERIC-
l’CR (Fig. 2C) protocols were used. as accentuated by the
arrowheads in Fig. IB and C. lanes IS

The second group (group B). inclttding the rcl‘erence strain
[or the patltov' tr (ATCC 35037) originally isolated in Ne\\
Zealand lw D W. Dye in 1955 (Fig... ‘. lanes 15). and strain
ATCC 11551 isolated' 1n 11)-'1? in lndiana (lattes 1(1). appeared
to have identical ﬁngerprint patterns but were \ery distinct
from isolates in group A. isolates in groups A and B appeared
to share only one single band ofequal mobility. as generated by
the ERlC- PCR and highlighted with an arrowhead itt Fig. 2C
anes 111. Although 11 cannot be ruled out that this Lomigration
pattern is lottuitous itt tltis particular Lase such disparate
ﬁngerprint proliles may indicate cases in which two or tnore
evolutionary lines have converged to a similar pathogenic
phenotype and have been erroneously classiﬁed within the
same taxou.

Reproducibility of DNA ﬁngerprints. DNA ﬁngerprints for
speciﬁc strains could be consistently generated from a single
preparation 01’ DNA (data not shown). To determine the
reproducibility ol' generating ﬁngerprint proﬁles from ditferent
DNA preparations of the same strain. we analyzed DNA from
cultures initiated from single colonies attd extracted at ditl'er-
enl times (over a period 111' several months), An example of this
analysis. using ERIC-PCR. is shown in Fig. 4. Strains that
yielded some 01. the most complex ﬁngerprint patterns were
used for this purpose. and in each case identical proﬁles were
generated (compare lanes 1 and 3. lanes 3 and -l. etc.). The
single ERICPCR polymorphism noted among isolates of .\'.
(11111111110111 pv. pelargonii (Fig, 2(‘) had been of particular
interest. This polymorphism was reproducible (see the arm“-
ltead in Fig. -l. lanes 5 and (1 versus lanes 7 attd X).

To determine it whole cells. as opposed to puriﬁed DNA.
could be used for rep-PCR. we compared the rep—PCR pat—
terns resulting l’rom the two protocols, The qualit} of DNA

 

.\i-t\7‘1/111/01V‘11‘ ANI) I'SI-‘I'DUJIUA',IS woven t-‘INGERPRlNTs

.‘I‘H

812345678910118

 

FIG Comparison ofwhole-cell rep- P—CR with rL'p PCR 111whiclt
puriﬁed genomic DNA nus used. A n- old-pl portion ol each of the
rep-PCR mixtures was loaded onto 11 MW agarose gel. The resulting
REP-PCR electropltoretic patterns generated l’to111..\’ 11111111111111 pv
\esicatoria P 03 DNA (lane I) or 1 pl 01 a cell suspension itt watet
(lanL L‘ 2):P..r1'r111u111:p\. tomato 915 DNA (latte .1) 1r 11d ol 11 eL\ll
suspension esudate from a lesion on 11 tomato leaf (lanL J):\ ..
1'11111/11- 1111.1 pv. pelargonii ill} DNA (lane 51.1 pl of a cell suspension
c\ud1111: 1111111 a lesion on a geranium leat (lane ()1. or cells collected
directly tront solid media (lattes 7 11nd \‘1; 11nd BOX— PCR ploducts
gLnet'ated lrom .\ 1.1111111111111- p\.pelargon1i1\'03 DN. \ (lane ”1111 trom
cells collected directly t'rom solid media (lanes to and 11) are shown.
Other details are described in the legend to Fig. l

preparations did not alteet ﬁngerprint proﬁles substantially.
since whole cells yielded similar proﬁles to those generated
from isolated DNA (Fig. 5). Analogous REP~PCR and BOX-
PCR genomic ﬁngerprints were generated from puriﬁed
genomic DNA and from cells suspended in nater. derived from
cultures on solid media. or cwded from plant lesions Figure 5
shows the leP- PCR ptoﬁlc generated lrom DNA of .\'.
11111111111111 pv. vesicatotia (latte l) isolate (P413) compared with
the proﬁle generated from cells suspended in water (lane 2).
Lanes 3 and 4 show tlte ﬁngerprint generated front DNA of P.
n'ringm' pv. syringae 015 (lane 3) compared with the proﬁle
generated from cells collected from 11 plant lesion (lane 4).
Likewise. lanes 5 through 8 show the REP- PCR pattern
generated flom DNA of \’ ca111pc..cm'r p\. pelargonii 803 (latte
51 cotttpzued with the proﬁle generated lrom cells collected
from a plant lesion (lane (1) or from cells collected from solid
media (lattes 7 and 8). Positive diagnosis was achieved within
Z-l l1 if 11 proﬁle could be gettetated directly from infected plant
tissue or after I to J days it. bacteria were ﬁrst cultured on 11
solid medium. Several tomato and geranium plants collected
from commercial greenhouses or submitted to the Michigan
State University plant-diagnostic laboratoty were evaluated in
this manner to 1-11pidlt- identify the causal org oanism. Finally.
two bacterial culttues on solid media and thought to be .'.\
cuntpcxlm pv. pelatgonii yielded BO.\ PCR proﬁles (Fig.1 5.
anes it) and ll) identical to those ot 11 known .\. 11111111111171 p\
pelargonii isolate (SIB [lane 9]) Positne Lottﬁtntatiou of the
identin ol the isolates was achieved witltin l-l it of sample
subtttission.

DISCUSSION
In this study we have demonstrated that repetitive extragenic
sequences such as REP. ERIC. and BOX are present in the
genome ol’ diverse .\‘11111/1111111111115 and Pscmlontonns strains.

 

 

 

217

22‘): L0 U WS ET AL.

conﬁrming and extending the conclusion of Versalovic et al.
(63). de Bruijn (7). and Koeuth ct al. (32) that these sequences
are virtually ubiquitous. We have also demonstrated that the
REP-. ERIC —. and BOX-PCR protocols. referred to as rep-
PC R collectively. are particularly suitable for the rapid molec-
ular characterization of plant-pathogenic bacteria. especially at
the pathovar level. The rep-PCR protocol clearly has the
potential to differentiate pathovars. including those that are
not easily distinguished by other phenotypic and phylogenetic
techniques. rep-PCR may also be capable of discerning
whether pathovars represent a single evolutionary line or are
composed of several lines that have converged to exhibit a
similar pathogenic phenotype.

The data presented here suggest that rep-PCR should also
be a useful tool for diagnostic purposes in plant pathology.
Similar suggestions have been made about the utility of
rep-PCR in infectiousdisease diagnosis and epidemiological
analysis in human pathology (62. 64. 67). Several criteria must.
of course. be met if rep-PCR is to be useful for the proper
identiﬁcation of unknown isolates. First. the characteristic
location of REP-. ERIC-. and BOX-like sequences. and there-
fore the genomic ﬁngerprint patterns generated by rep.PCR.
must be stable over time and distance. Our results suggest that
this is the case. For example. the ATCC 11633 isolate of X
campestris pv. vesicatoria group A was ﬁrst isolated in the late
1940s. and isolates obtained from Taiwan. Mexico. and various
locations in the United States over the last several years (Table
l) have nearly identical ﬁngerprint proﬁles (Fig. 2). Likewise.
we have analyzed isolates of X. campestris pv. vesicatoria
collected in 1993 during epidemics in Michigan that have a
similar proﬁle to the X. campesm's pv. vesicatoria group B
isolates (Fig. 2). which date back to 1943 and 1955 (data not
shown). Comparison of the genomic ﬁngerprint proﬁles of
other isolates within a pathovar, separated by time or distance
(Table 1). supports the notion that the proﬁles remain stable.
The similarity of ﬁngerprint proﬁles from related isolates
separated by time has been noted by others (67).

Second. the rep-PCR technique must be able to discriminate
among related but distinct bacterial strains with sufﬁcient
resolution and be reproducible. We have demonstrated that
closely related bacteria, as determined by other experimental
protocols such as DNA hybridization studies. can have diver-
gent rep-PCR proﬁles and that bacteria associated with the
same host or disease complex can be easily differentiated
because of the complexity of DNA ampliﬁcation products
generated by using the three diﬁerent primer sets. Identical
ﬁngerprint proﬁles generated from DNA isolated from single-
colony cultures of an isolate (Fig. 4) and from isolates sepa-
rated by time or distance (e.g.. X. campestris pv. pelargonii [Fig
2]) support the reproducibility of the rep-PCR protocol.

Third. background work with large numbers of isolates will
have to be done to ascertain the homogeneity or heterogeneity
of ﬁngerprint proﬁles within each pathovar.

We systematically compared the general utility of REP.
BOX. and ERIC primers (Fig. l to 3). In general. differences
between pathovars and substantial polymorphism (dissimilari-
ties between strains within a pathovar) were detected by each
primer set with similar resolution. suggesting that the distribu-
tion of REP. ERIC. and BOX sequences is a true reﬂection of
genomic structure. For detecting limited polymorphisms within
a clonal group or apparent similarities between pathovars.
each primer set olfered unique information. For example,
REP-PCR suggested that X. campestris pv. poae and pv.
graminis were closely related, whereas this was less obvious
with ERIC-PCR. The REP- and ERIC-PCR ﬁngerprints of
isolates of X. campestris pv. campestris were highly diverse. but

 

 

Arm. Esvmox. Mlt‘komot.

analogous proﬁles were more obvious when BOX-PC R was
used. By using three different primer sets. a broader survey of
the chromosomal structure was possible and more speciﬁc
conclusions concerning diversity or similarity among strains
and pathovars were achieved.

Reproducible ﬁngerprint proﬁles of a particular strain can
be generated from DNA isolated at different times from the
same colony. from different colonies of the same strain. or
from serial cultures of the same strain (47: also see above).
However. the rep-PC R protocol is subject to the same exper-
imental caveats as all other PC R-based techniques. such as the
problem of amplifying even minute amounts of contaminating
DNA. variations in template and primer DNA concentrations.
and the composition of the PCR buffer. Moreover. the elec-
trophoresis conditions can inﬂuence the interpretation of
results. Therefore. we recommend that the rep-PCR be carried
out at least two independent times with appropriate negative
controls (e.g.. no template DNA in the reaction mixture and/or
no primer DNA) and that the rep-PCR products be separated
on at least two independent gels (47).

In this study. a limited amount of variation in rep-PCR
patterns. probably as a result of the above limitations. was
observed. For example. more faint bands generated from DNA
of the P. syringae pv. syringae strains with ERIC-PCR were
observed in Fig.5 (lanes 9 to 16) than in Fig. 3C (lanes 4 to 7).
In general. depending on the efﬁciency of the PCR. large bands
(greater than 5 to 6 kb) and faint bands were not always
ampliﬁed to the same extent or were not equally visible after
ethidium bromide staining. More prominent bands. however.
were consistently present and were almost always sufﬁcient to
identify speciﬁc pathovars or strains and able to distinguish
them from closely related strains.

Other PCR-based genomic ﬁngerprinting techniques have
been described recently. For example. arbitrary primers and
PCR (65, 66). also known as random ampliﬁed polymorphic
DNA (RAPD) analysis. has a demonstrated use for diﬁeren-
tiating bacteria and for diagnostic purposes (6. 13). There are
some crucial dilferences between rep-PCR and RAPD-type
analyses that are particularly relevant when considering the
reproducibility of diagnostic protocols. The major difference
lies in the length of the primers used and the corresponding
PCR conditions. RAPD analysis relies on the use of primers
with arbitrary sequences (most commonly loomers but up to 34
bp [65. 66]). whereas rep—PCR involves the use of primers of 18
to 22 bp with high homology to repetitive sequences (32. 63).
The latter primers allow the use of more stringent PCR
conditions, which, in turn may reduce experimental variation
and PCR artifacts.

In this study. we also show that it is not necessary to use
puriﬁed genomic DNA as a template for rep-PCR. Bacterial
cells derived from single colonies on agar plates or from liquid
cultures or eluted directly from pathogen-induced lesions on
plants could be used directly for rep-PCR. Apparently, the
cycling of the reaction at 95°C released enough DNA from the
cells to serve as a template for rep—PCR. In fact. rep-PCR with
bacterial cells yielded genomic ﬁngerprinting patterns indistin-
guishable from those generated with puriﬁed template DNA.
This phenomenon has been observed with a variety of gram-
negative and gram-positive bacteria (47. 68) and with plant
(nodule) tissues infected with symbiotic Rhizobium, Azorht'zo-
bium. or Bradyrhizobium isolates (45. 47). This form of “whole-
cell rep—PCR” (68) is particularly useful for rapid and routine
diagnostic analyses.

Reproducible and routine rep-PCR-based genomic ﬁnger-
printing for the identiﬁcation of phytopathogenic bacteria is
expected to be further facilitated by the use of ﬁuorescently

 

 

218

VOL. 60. I994

labeled REP. ERIC. and BOX primers and the Applied
Biosystems DNA Sequenator equipped with the Genescan
software package. as has been demonstrated for human DNA
analysis using ﬁuoresccntly tagged short tandem repeats (14).
Preliminary results suggest that the generation of “bar code-
like" genomic ﬁngerprints of bacteria and their storage in data
ﬁles by this method is possible (39. 61).

Although the rep-PCR technique is very useful for bacterial
strain identiﬁcation. the utility of REP-. BOX-. and ERIC-
PCR for bacterial taxonomy may be limited to closely related
strains. as noted by others (7. 28. 67). For assessing the genetic
distance between diverse bacteria. rRNA gene-based tech-
niques are likely to be more useful (5. 25). Conclusions about
evolutionary relatedness by rep-PCR. especially on the basis of
a few comigrating bands. would have to be conﬁrmed by DNA
sequencing or hybridization analysis. In this study. few com~
mon bands among pathovars. even those known to be closely
related. were observed. Of particular interest were the two
pathovars of X. oryzae (Fig. 1. lanes 1 and 2). X. oryzae pv.
oryzae and pv. oryzicola have a DNA hybridization value
greater than 90% (52) and cannot be reliably distinguished in
129 of 133 phenotypic tests (60). However. each pathovar has
specialized to incite unique symptoms on rice plants (42). This
specialization may be correlated with the distribution of REP.
BOX. and ERIC sequences to the extent that a conclusion of
common ancestry cannot be made on the basis of the resultant
ﬁngerprint proﬁles. Approaches other than pathogenicity tests.
such as protein proﬁle analysis or fatty acid proﬁle analysis
(59). serologic testing (3). and rRNA gene restriction patterns
(5). support the distinctiveness of X. oryzae pv. oryzae and pv.
oryzicola.

X. campestris pv. poae and pv. graminis (Fig. 1. lanes 3 and
4) offer another unique comparison. These pathovars. includ-
ing the strains used in our study, have similar fatty acid proﬁles
(59) and cannot be distinguished in 215 of 257 phenotypic tests
(53). However. the pathovars have distinctive sodium dodecyl
sulfate-polyacrylamide gel electrophoresis proﬁles of whole-
cell proteins and are accepted as distinct pathovars. The
primary criterion for Splitting the pathovars is based on host
range studies (12). The rep-PCR ﬁngerprint proﬁles between
the two pathovars contain many bands of equal mobility. but
they are distinct. consistent with the concept that selection for
a specialized niche affects genome organization (33) and that
this corresponds to a unique distribution of repetitive se-
quences in the bacterial genome.

The rep-PCR technique may be limited for phylogenetic
analysis. but it effectively differentiated two evolutionary lines
classiﬁed within the same taxon of X. campestris pv. vesicatoria.
Two distinct groups have also been noted by Vauterin et al.
(57) and Stall et al. (50).

In conclusion, we have found that REP-. BOX-, and ERIC-
like sequences are prevalent in strains of X. oryzae. X. campes-
tris. and P. syringae and can be exploited to generate genomic
ﬁngerprints of strains within each species. Selection for a
specialized niche appears to affect the distribution of repetitive
sequences. resulting in ﬁngerprints unique to speciﬁc patho-
vars or strains. Unique ﬁngerprint proﬁles generated by rep-
PCR can be exploited for diagnostic purposes and for discem-
ing evolutionary lines that make up a pathovar. Disclosing the
population diversity of each pathovar, in turn. has implications
for the implementation of breeding programs, other disease
management strategies. and ecological and epidemiological
studies.

 

.\Bl:\’THO:II().\'.-l.§ AND PSEL'I)().\I(),\.-I.S rep-PCR FINGERPRINTS 3293

ACKNOWLEDGMENTS

This study was supported tn part by a scholarship from the Natural

Science and Research Council of Canada and a C. S. Mott Predocmral
Fellowship-MSU (to FJ.L.). FJ.D. gratefully acknowledges support
from the DOE (grant DE-FG()2-9IER2lltl21) and the NSF Center for
Microbial Ecology (grant NSF-BlR-‘lllllllllol.

We thank Andre“ Jarosz for critical reading of the manuscript:

Maria Schneider for providing protocols and technical advice: and
Thearith Koeuth. Jim Versalovic. and Jim Lupski for providing
primers. unpublished data. and helpful discussions. We thank Karen
Bird for help in preparing the manuscript and Kurt Stepnitz and
Marlene Cameron for preparing the ﬁgures. We also thank the many
people (listed in Table I) who provided bacterial cultures or DNA.

.-

IJ

REFERENCES

. Aehtman. M. and G. Pluschke. I986. Clonal analysis of descent

and virulence among selected Escherichia coli. Annu. Rev. Micro-
biol. 40:185-2ll).

. Ausubel. P. M.. R. Brent. R. E. Kingston. D. D. Moore. J. G.

Seidman. J. A. Smith. and K. Struhl. 1992. Current protocols in
molecular biology. vol. I. Greene Publishing Associates and Wiley-
lnterscience. New York.

. Benedict. A. A.. A. M. Alvarez. .I. Berestecky. W. lmanaka. C. Y.

Mizumoto. L W. Pollard. T. W. Mew. and C. F. Gonzalez. I989.
Pathovar speciﬁc monoclonal antibodies for XamItomamts
campestris pv. oryzae and for Xantltomonus campcsms pv. oryzi-
cola. Phytopathology 79:322-328.

. Benedict. A. A.. A. M. Alvarez. and L. W. Pollard. I990. Pathovar

speciﬁc antigens of Xamlromauas campestris pv. begoniae and
Xanthomona: campestris pv. pelargonii detected with monoclonal
antibodies. Appl. Environ. Microbiol. 56:572-574.

. Berthier, Y.. V. Verdier. .I. Guesdon. D. Chevrier. J. Denis. G.

Decoux. and M. Demure. I993. Characterization of Xanthomonas
campestris pathovars by rRNA gene restriction patterns. Appl.
Environ. Microbiol. 59:851—859.

. Cancllla. M. R... I. B. Powell. A. J. Hillier. and B. B. Davidson.

1992. Rapid genomic ﬁngerprinting of Lactococcus lactis strains by
arbitrarily primed polymerase chain reaction with p32 and ﬂuo-
rescent labels. Appl. Environ. Microbiol. 58:l772—l775.

6a.Coo|tsey. D. A.. and .I. II. Graham. 1989. Genomic ﬁngerprinting

of two pathovars of phytopathogenic bacteria by rarccutting
restriction enzymes and ﬁeld inversion gel electrophoresis. Phyto-
pathology 79:745-750.

. de Bruijn. F. J. 1992. Use of repetitive (repetitive extragenic

palindromic and enterobacterial repetitive intergeneric consensus)
sequences and the polymerase chain reaction to ﬁngerprint the
genomes of Rhizobium meliloti isolates and other soil bacteria.
Appl. Environ. Microbiol. 58:2180-2187.

7a.Denny. T. P. 1988. Differentiation of Pseudomonas syringae pv.

12.

I3.

tomato from Rs. pv. syringae with a DNA hybridization probe.
Phytopathology 78:] 186—1 I93.

. Denny. T. P.. M. N. Gilmour, and R. K. Selander. 1988. Genetic

diversity and relationships of two pathovars of Pseudomonas
syringae. J. Gen. Microbiol. 134:1949—1960.

. Dunbar, K. B. and C. 1'. Stephens. 1989. An in vitro screen for

detecting resistance in Pelargom'um somaclones to bacterial blight
of geranium. Plant Dis. 73:9l0—912.

. Dye. D. W. 1962. The inadequacy of the usual determinative tests

for the identiﬁcation of Xanthomona: spp. N. Z. J. Sci. 5:393—416.

. Dye. D. W.. J. F. Bradbury. M. 6010, A. C. Hayward, R. A. Lelliot.

and M. N. Schroth. 1980. International standards for naming
pathovars of phytopathogenic bacteria and a list of pathovar
names and pathotype strains. Rev. Plant Pathol. 59:153-168.
Egli, T.. and D. Schmidt. 1982. Pathogenic variation among the
causal agents of bacterial wilt of forage grasses. Phytopathol. Z.
[04:138—150.

Feltete. A. J. A. Booth. 8. M. Halllng. and R. W. Stleh. I992.
Ampliﬁcation fragment length polymorphism in Bmcella strains by
use of polymerase chain reaction with arbitrary primers. J. Bacte-
riol. 174:7778-7783.

. Frégeau. C. J., and R. M. Foomey. 1993. DNA typing with

ﬂuorescently tagged short tandem repeats: a sensitive and accurate
approach to human identiﬁcation. BioTechniques 15:100—119.

 

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219

229.1 LOUWS ET AL.

". Gabriel. D. W.. and R. de Fuyter. I992 RFLP anahsis and gene

tagging for bacterial identiﬁcation and taxonom_\_ p st—o 1.1" S J
Gurr M J. McPherson and D. J. Bo “Ies (ed). Molecular plant
patholog\: a practical approach \ol I. IRL Press. New \

.Gabri 2.1 D. W J E. nter. M. T. Kingsley. J.“ .Miller
G. R. lam. I988 Clonal population structure of Aunt/Ionian":
campestris and genetic diversity among citrus canker strains. Mol
Plant- Microbe, Interact. I: 59

. Gabriel. D.“ .T. Kingsley. J. E. Hunter. and T. Gottwald.
I989. Reinstalemlent of A’umliumnnm curl (ex Hasse) and A.
plIﬂJt'Ull (ex Smith) to species and reclassiﬁcation oI all A. campu-
Iri.\ p\ ciIrI strains Int. J Syst. Bacteriol 3.

. (.Ilson. E..J M. CIe’ment.D. Brutlag. and M. Hofnung. 198-l. A
famih of dispersed repetitive extlr7age4nic palindromic DNA se~
quences in E cuII. EMBO J. 3:I

.Gilson. E.. J. .Clément. D. Ple7rrin. M Hofnung. I987.
Palindromic units: a case of highly repelitnive DNA sequences in
bacteria Trends Genet. 3'- 726— 6—30.

. Hartu ung. J. S. 1992. Plasmid- based hybridization probes for
detection and identiﬁcation of Xantliomonm campcsn-Ii pv. citri.
Plant Dis. 76: 889-893

. Hart rtung ..I. 5.. and E. L. Civerolo. I987. Genomic ﬁngerprints of
Aamlrmnonars dcampesmx pt. citri strains from Asia. South Amer-
ica. an dFl da. Phytopathology 77:282—285.

_. Higgins.C. OF. G. F.A ' a.rnes J. M. Clement. and M.

Hofnung. 1982 A novel intercistronic regulatory element of

Palleroni. and M. N. Schmth. I990.
Deoxyribonucleic acid related es of 24 xanthomonad strains
representing 23 Xanlhomonas campestnr pathovars and XIII
Ilromonar fragamu'. J. App LB acleriol. 68:26
ton. C. S. J. C. F. Higgins. andP.M. Sharp. 1991. ERIC
sequences: a novel family of repetitive elements In the genomes of
Escherichia coli. Salmonella n'p/Iimun'um and other enterobacteria.
Mol. Microbiol. 5:81
. Jensen. M. A.. J. A. Webster. and N. Straus. 1993. Rapid identi-
ﬁcation of bacteria on the basis of polymerase chain reaction—
ampliﬁed ribosomal DNA spacer polymorphisms. Appl. Environ.
MicrobioL 59:9 45—
J. B.. R. D t"Gitaitis. and S. M. McCarter. I986. Fluores‘
cence on single-carbon sources for separation of Pseudomonas
syringae pv. syr yringac. RM .to and P. viridiﬂava on
tomato transspliqnts. Plant Dis. 70:151—153.o
Jones. J. 3.. McCarter. and R. D. Gitaitils. 1981. Association
of Pseudomosnassv syringae pv. syringae with easf spot dIsease of
tomato transplants In southern Georgia Phytopathology 71: 1281—
1285.

Judd, A. K.. M. Schneider. M. J. Sadows try. and F. J. de Bruijn.

1993. Use of repetitive sequences and the Iymer rase chain

reaction technique to classify genetically related Bradyrhizobium

japonicum serocluster 123 strains. Appl Environ MicrobioL 59:

1702—1708.

Kamoun. 5.. and C. 1. Katie. 1990. A plant- -inducible gene of

Xanthomona: campestris pv. campestris encodes an exocellular

component required for growth in the host and hypersensitivity on

nonhosts. J. Bacteriol. I72: 5165-51 7.

King. E. 0.. M. K. Ward. and D. E. Raney. 1954. Two simplbe

media for the demonstration of pyocyanin and ﬂuorescin. 1.1.2

Clin. Med. 44'

. King. G. J. 1989. Plasmid analysis and variation in Pseudomonas
syringae. J Appl. eBacterial 67:4 89-4 96.

Koe uth. T.. rsalovic. and J. R. Lupski. Diﬁerential subse-
uence conservation supports the mosaic nature ofin terserspe

repetitive BOX elements In bacteria. Submitted for publication

1.985 Minireviewo. Concept of a bacterial species Int.

.1. Syst. Bacteriol 35' 217—22

. Kn' 5.. and M. Riley. 0I990 Organization of the bacterial
chromosome. Microbiol. Rev. 54:502—53

. Latorre m. B.A.. andA. LJones. I979. Pseudomonas marspmnomn I.

the cause of bacterial canker of sour cherry in Michigan. and its

epiphytic association with P. mingalc. 8Phytopathology 69:335— 339

Law. G. R.. an nd D. W. G 7.eronse ation of plasmid

DNA sequences and pathovar identiﬁcation of strains of Xan-

53. Van Den Mooter. M., M. Stee

56. Vauterin,

AI'I’L EV\ IRO\. MICROBIUL.

Ilmnmnm tanI/n' rim. Phytopathologx 77: 448—I5?
37. Iazo. . R.. R R If Gabriel. I987. Pathmars of
Amnlromuuus cum/urinal are distinguishable b\ restnction fran-
J. Syst. Bacteriol, 37uzl2'I4—221.

.1990. A
repetitiw DNA sequence diﬁerenttntes A'IIIIIltoanIIunn Irilmpcum
p\. oryzae from other pathma rs ol A r:lIIIpt'sIn1.Mol lant—

Microbe Interact. 3. 238— l-lb.

39 Louws. F. J.. and dF. J. de Bruijn. Unpublished data.

41). Lupski. J. K.. and G. M. Weinstock. 1992. Short. interspersed
repetitixe DNA sequences in prokaryotic genomes. J. Bacteriol
174:452 '4529.

41. Man nin. 11.. 0. Humbert. M. Camara. E Guenzi. J. Walker. T.

Mitchell. P. Andrew. Pru homme. G. Alloing, R. Hakenbeck.

rnson. G. J. Boulnois. andJ. -P. CIaven-s. 1992, A highly

cons served repeated DNA element located In the chromosome of
Streptococcus pnctmmniut'. Nucleic Acids Res. 20: 3479— 3483.

42. Mevi ..T W- M. Alv a-rezJ Bleach. andJ. Swings. 1993 Focus
on bacterial blight9 of rice. ePlant Dis.

43. Palle croni. N. J. nus I.P.tct71d0n_wuas Migula1894. p.
141— 198.1" J. G. Holt and N. R. Kreig (ed.). Bergey‘s manual of
systematic bacteriology vol I. The Williams & Wilkins Co..
Baltimore

43a.Paterson. J. M.. and A. L Jones. 1991. Detection of Pseudomonas
n'n‘nguc p\. morsprunorum on cherries in() Michigan with a DNA
hybridization probe. Plant Dis 75' 893-8

44 Pecknold. P. C.. and R. G. G 93. eoxyribonucleic acid
homology groups among phytoplathogenic Pseudomonas species
Int. J. Syst. Bacteriol. 23:11 1—12

45. Sadowsky. M.. M. Schneider. and F. J. de Bruijn. Unpublished

46, Sambrook. J.. E. F. Fritsch. and 'I‘. Maniatis. 1989. Molecular
cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Labo—
ratory Press Cold Spring Harbor.N

47. Schneider, M.. and F. J. de Bruijn. Unpublished data.

48. Selander. R.K.. an d.J M. Musser. 1990. Population genetics of
bacterialpathoge es is p. 11—36. In B. H. IglewskI andV L. Clark
(e e.d ). Mol ular basis of bacterial pathogenesis. Academic Press.
Inc.. San Diego. Cai

49. Sharples. G. J.. and R. G. Lloyd. 1990. A novel repeated DNA
sequence Iocatedn In the intergenic regions of bacterial chromo-

so.mes Nucleic Acids Res. 18: 605 3—6508.

50. Stall. R. E.. C. Beaulieu. D. Egel, N. C. Hodge.I R. P. Leite, G. V.
Minsavage. H. Bouzar. J. B Jon ,A. M. A.
94 Two genetically diverse groups of strains are
included in a patho var rof Xanthomona: campestﬁ1.1m...l Syst.

Bacteriol. 44:47— 53.

51. Stead. D.E. 1992. Grouping of plant- pathogenic and other Pseudo-
monas species by using cellular fatty acid proﬁles. Int. J. Syst.
Bacteriol. 42:128— 95.

52. Swings. J..M Van Den Mooter. L Vauterin. B. Hoste, M. Gilles.
T. W Mew. and K. Reuters. 1990. Reclassiﬁcation of the causal
agents 01' I, , pv oryzae and
bacterial' " ' ‘ ' ' Micnla) (If rice
as pathovars of Xanthomona: oryzae (erx Ishiyama 1922) sp. nov. .,
nom. rev. Int. J. Syst. Bacteriol. 40:309— 11.

nacke rs, C. Mnertens. F Gossele, P.

De Vos. J. Swings. K. Kersters, and J. De p"Icy. I987. Differentia-

tion berwee nXmI Ihomonas campeslm pmgraminis (ISPP List

1980) pv.i phleip7ratensis (ISPP List 1980) amends pv poae Egli

and Schm 82 by

numerical analysis ofri phenotypic features alnd protein gel 1eglectro-
phoregrams. J. Phytopathol. II8:135—I

Van

 

54. Dan Moo ter M.. andJ. Swings. I99I). Numericalanalysis of

295 phenotypic features of 266 Xanthomona: strains and related
strains and an improved taxonomy of the genus. Int. J. Syst.
Bacteriol. 40:348—369.

55. Van Zyl. E- and P. I- Steyn I990. Differentiation of phytopatho-

genie Pseudomonas and Xanthomona: species sand pathovars by
uem ritcal axon omy and protein gel electrophorcgrams. Syst.

Appl. Microbiol. I3:60—7I.

J. Swings, and K. Kersters. 1991. Grouping of

Xanrliomanas campestris pathovars by SDS-PAGE of proteins. J.

 

V111,. 611. I994

J1

'JI
ft

'JI
C

5‘

3‘

IJ

220

Gen. Microbiol. 137' 1677—1687

.7. Vauterin. L..J. Swings. K. Kersters. M. Gilles. T. W. Men. M. N.

chmth N. J. Palleroni D. C. Hil Idebrand. D. E. St lac a.d E. L.
Civerolo. A. C. Hayward. H. Marnilc. R. E. Stall. A. K. Vidaver.
radbury. I991) Tovlard an improwd taxonomy ol
AIIIII/mnmnm. Int. J. Syst. Bacteriol. 40: 312- 316
. Veau erin. R. Vantomme. B. Pot. B. Host e..J Swi rugs. and K.
Kensers. I990 Taxonomic analysis of AmIIlIonIOInIIIs Donut/Morris p\
hegoniac andA Ca"lp(‘5!rl.\ p\ pelargonii b_\ me ophytopatho-
logical. phenotypic protein electrophoretic and" DNA hybridiza-
tion methods. Syst. Appl. Microbiol. 13:166—[7
. Vauterin. L. P. Yang. H. Hoste. B. Pol. J. Swings. and K'. Kersters.
I992. Taxonomy of xanthomonads from cereals and grasses based
on SDS-PAGE of proteins. fatty acid analysis and DNA hybrid-
ization. J. Gen. Microbiol. [38:1467—I477.
(I. Vern Cruz.C ...M F. Gossele. K. Kersters. P. chers. M. Van Den
win gs. and J. DeLc .1984. Diﬁerentiation between
Xantlmmonas cam srrix urXatI/Iomonas campestris pv.
mico ola a he bacterial brown blotch pathogen on rice b_\
numerical analysis of phenotypic features and protein gel electro-
phorcgrams. J. Gen. Microbiol. 130:2983-

. Versalovic. J.. V. Knpur. T. Koe G. Mazurek. T.S .

J. Musser. and J. R. Lupski. Automated DN Anﬁ gerprinting oi
pathogenic bacteria by ﬂuorophore-enhanced repetitive sequence
based PCR. Submitted for publiM at on.

Versalovic. J..V . Knpur. E. O.M n...Jr U.S huh. T. Koemh .
J. R. Lupski andJ M. Musse on 1993. nIi‘enicillin-resistantSlrcpilo-
Luccux pneuntoniac str ains recovered in Hou iend ticaﬁ on
and mo occl ular characterization of multiple clones. J. Infect. Dis.
167:850—856

11

. Versalovic. J.. C. R. Woods. Jr.. P

3‘

'JI

3‘
5‘

7‘

0‘
on

9

C

\I
O

AC4N'THOMONAS AND PSEL'I)().II(),\'.-IS rep-PCR FINGERPRINTS 2295

()3. Versalovic. J.. T. Koeuth. and J. R. Lupski. 1991. Distribution of

repetitive DNA sequences in eubacteria and application to ﬁnger

printing of bacterial genomes. Nucleic Acids Res. 192682341831.

R Georghiou. R. J. Hamill. and
R. Lupski. I993. DNA-based identiﬁcation and epidemmlogtc

typing of bacterial pathogens. Arch. Pathol. Lab. Med. 11711088-
I98.

Welsh.J.. and M. McClelland. I991]. Fingerprinting genomes using
PCR with arbitrary primers. Nucleic Acids Res. 18:7213-7218.

. Williams. J. G. K.. A. R. Kubelik. K. J. Livak. J. A. Rofalski. and

S. V. Tinget. 1990. DNA polymorphisms ampliﬁed b\ arbitran
primers are uselul as genetic markers. Nucleic Acids Res. I8: 6531—
65 3.".

.Woods.C R...J V'ersalovic.T. Koeuth. and J. R. Lupski. I992

Analysis of Arelationships among isolates of CitrolIacrr-r dII I'm/1 b_\
using DN Aﬁngerprints generated b_\ repetitive sequence-based
primers in the polymerase chain reaction. J. Clin. Microbiol.
30:2921—2929.

. Woods. C. R.. J. Versalovic. T. Koeuth. and J. R. Lupski. I993.

Whole-cell repetitive element sequence-based polymerase chain
reaction allows rapid assessment of clonal relationships of bacte-
rial isolates. J. Clin. Microbiol. 31:1927—1931.

. Young. J. M.. J. F. Bradbury. R. E. Dar/5.11. S. Dick '.G. L

revisions of plant pathogenic bacteria and list of names 19804988.
Rev. Plant Pathol. 70: 213-

. Young. J. M..Y .Takikawa. L Garden. and D. E. Ste ad. 1992

Changing concepts in the taxonomy of plant pathogenic bacteria.
Annu. Rev. Phytopathol. 30:67—105.

 

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