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Documenting and characterizing Phytophthora capsici from
irrigation water and bean in Michigan and screening for fruit
resistance in cucumber

presented by

Amanda Jane Gevens

has been accepted towards fulfillment
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DOCUMENTING AND CHARACTERIZING PHYTOPHTHORA CAPSICI
FROM IRRIGATION WATER AND BEAN IN MICHIGAN AND SCREENING
FOR FRUIT RESISTANCE IN CUCUMBER
By

Amanda Jane Gevens

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Plant Pathology

2005

ABSTRACT
DOCUMENTING AND CHARACTERIZING PHYTOPHTHORA CAPSICI
FROM IRRIGATION WATER AND BEAN IN MICHIGAN AND SCREENING
FOR FRUIT RESISTANCE IN CUCUMBER
By
Amanda Jane Gevens

The fungal-like oomycete Phytophthora capsici causes disease on cucurbit,
solanaceous, and more recently, leguminous crops worldwide. Surface irrigation sources
in three Michigan counties with a history of susceptible crop production were monitored
for P. capsici during four growing seasons. Pear and cucumber baits were used to detect
the pathogen. Recovered P. capsici isolates (270) were screened for sensitivity to the
fungicide mefenoxam and characterized for compatibility type (CT). Amplified
Fragment Length Polymorphism (AF LP) analysis of select isolates was carried out to
indicate the persistence of isolate Similarity over time and Space. Phytophthora capsici
was not found to overwinter in the eleven Michigan water sources monitored. The
consistent detection of P. capsici in surface water used for irrigation in Michigan
suggests that this is an important means of pathogen dissemination.

Historically, beans (Phaseolus vulgaris) have been considered a suitable crop for
rotation with P. capsici-susceptible crops. Commercial bean fields in three Michigan
counties exhibited water-soaked foliage, stem necrosis, and overall plant decline along
the surface water drainage pattern. All fields had a history of P. capsici infestation.
Diseased tissue from stems, petioles, leaves, and pods collected yielded a total of 680

isolates of P. capsici. No isolates were recovered from bean roots. Under laboratory

conditions, representative bean isolates were pathogenic on cucumber fruit and twelve

different types of bean, including soybean. AF LP analysis was carried out to investigate
the genetic diversity among isolates and geographical populations. There was no
subdivision in field populations of P. capsici based on host or plant tissue. This is the
first documentation and etiological report of P. capsici on bean in Michigan. At this
time, rotating beans with other susceptible hosts is not recommended.

Cucumber fruit rot, caused by P. capsici, has become a persistent threat in most
commercial production regions. Although mature cucumber plants exhibit some
resistance to disease by P. capsici, the fruit are particularly susceptible. Identification
and utilization of resistance in cucumber fruit would provide a viable disease
management strategy for producers. The objectives of this study were to develop a
screen for testing detached cucumber fruit for resistance to P. capsici and to screen
cucumber cultigens for resistance. This is the first report using an unwounded fruit
screen to analyze cucumber resistance to P. capsici. Although no fruit exhibited
complete resistance to the pathogen, some cultigens showed limited pathogen

sporulation.

ACKNOWLEDGEMENTS

I am grateful for the guidance and encouragement of Dr. Mary Hausbeck, my
major professor. Her dedication and commitment to sound research and extension have
been an extraordinary example.

Many thanks to Brian Cortn'ght and Sheila Linderman for their technical assistance
in the field and in the laboratory. I appreciate all the assistance and friendship provided
by the following individuals: Jeff Woodworth, Kyle Cervantes, Shaunta Hill, Ryan
Bounds, Buck Counts, Blair Harlan, Dr. Catarina Saude, Bryan Webster, Jacob Witer,
and Andrew Worth. Additional thanks to my committee members, Drs. Ray
Hammerschmidt, Annemiek Schilder, Mathieu Ngouajio, and Jan Byrne for their support
and critical analysis of my work.

Last but not least, I am grateful for the love and support of the Gevens and Jordan
families. To my husband, Stephen Jordan, your academic and personal support have

been tremendous. You will always be my favorite plant pathologist.

iv

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................... vii
LIST OF FIGURES ....................................................................................................... ix
LITERATURE REVIEW
Introduction ........................................................................................................... 2
Pathogen Life Cycle .............................................................................................. 3
Pathogenicity and Virulence ................................................................................. 9
Baiting for Phytophthora spp .............................................................................. l 1
Management ........................................................................................................ 14
Literature Cited ................................................................................................... 23
CHAPTER I

Baiting Phytophthora capsici from Michigan surface irrigation water and
characterization of isolates

Abstract ............................................................................................................... 37
Introduction ......................................................................................................... 38
Materials and Methods ........................................................................................ 40
Results ................................................................................................................. 47
Discussion ........................................................................................................... 64
Acknowledgements ............................................................................................. 69
Literature Cited ................................................................................................... 70
CHAPTER 11

Identification and characterization of Phytophthora capsici on bean (Phaseolus
vulgaris) in Michigan

Abstract ............................................................................................................... 74
Introduction ......................................................................................................... 75
Materials and Methods ........................................................................................ 77
Results ................................................................................................................. 83
Discussion ......................................................................................................... 1 02
Acknowledgements ........................................................................................... 1 06
Literature Cited ................................................................................................. 107

CHAPTER III
Development of a detached cucumber (Cucumis sativus) fruit assay to screen for
resistance to infection by Phytophthora capsici

Abstract ............................................................................................................. 11 1
Introduction ....................................................................................................... 1 12
Materials and Methods ...................................................................................... 1 14
Results ............................................................................................................... 122
Discussion ......................................................................................................... 1 25
Acknowledgements ........................................................................................... 1 27
Literature Cited ................................................................................................. 128
APPENDIX A ............................................................................................................. 131

vi

LIST OF TABLES

CHAPTER I

Table 1. Time of sampling and P. capsici detection for SW Michigan

(Allegan County) river sites, 2002 to 2005 .................................................... 41
Table 2. Time of sampling and P. capsici detection for NW Michigan

(Oceana County) pond sites, 2002 to 2005. ................................................... 42
Table 3. Time of sampling and P. capsici detection for Michigan

ditch sites (Lenawee and Oceana C03), 2002 to 2005. ................................. 43
Table 4. Summary of P. capsici baiting isolate characterization for

SW Michigan (Allegan County) river system, 2002 to 2005. ....................... 51
Table 5. Summary of P. capsici baiting isolate characterization for

NW Michigan (Oceana Co.) ponds, 2002 to 2005 ......................................... 52

Table 6. Summary of P. capsici baiting isolate characterization for

Michigan ditches (Lenawee and Oceana C05), 2004 and 2005. ................... 53

Table 7. Average water temperature (°C) during baiting periods
from all sites and years (2002 to 2005) and the number of baiting

periods with positive detection of P. capsici. ................................................ 56

CHAPTER II
Table 1. Bean types included in laboratory P. capsici pathogenicity

screen. ............................................................................................................ 82

Table 2. Phytophthora capsici isolates identified from commercial
beans in Michigan fi‘om 2003 to 2005. Compatibility type (CT)

and mefenoxam sensitivity are characterized for each isolate. ...................... 88

Table 3. Analysis of P. capsici isolates identified from commercial
bean fields in 3 Michigan counties during 2003 to 2005 by specific

plant tissues. No P. capsici was identified on root tissue. ............................ 89

Table 4. Bean plant tissue diversity of AF LP clonal groups of
P. capsici isolates from Oceana and Cass Cos. Michigan in

2003 and 2004. ............................................................................................. 100

vii

CHAPTER HI
Table 1. Cucumber cultigens screened for fruit resistance to
P. capsici. .......................................................................................................... 118

Table 2. Re-screened cucumber culti gens which exhibited reduced
pathogen sporulation under fruit inoculation with P. capsici, 1999

to 2005. ........................................................................................................ 120
APPENDIX A
Table A. 1. Cucumis sativus cultigens screened for fruit resistance
to P. capsici during 1999 to 2005. ............................................................... 131

viii

LIST OF FIGURES
(Images in this dissertation are presented in color)

LITERATURE REVIEW
Figure 1. Disease cycle of P. capsici on pickling cucumber. Sexual
(oospores) and asexual (sporangia and zoospores) spores of P. capsici
in association with a pickling cucumber host. Oospores are produced
when both A1 and A2 compatibility types come into contact ......................... 4

Figure 2. Ultrastructure of a P. capsici oospore. The antheridial
wall remains attached to the oospore after fusion of the oogonia
and antheridia. The average oospore diameter is 20 um. Labeled
oospore components include: ooplast, lipids, oosphere, inner
and outer oospore walls, periplasm, oogonia] wall, and
antheridial wall ................................................................................................. 7

CHAPTER I -
Figure 1. A) Baiting station for P. capsici and contents: two green pears,
one cucumber from an uninfested source, and a temperature sensor.
B) Baiting station deployed in a pond used for crop irrigation.
C) Cucumber bait retrieved after one week in water monitoring site.
D) Pear bait retrieved after one week in water of monitoring site. ................ 44

Figure 2. Schematic of six sites along a river and creek system of SW
Michigan which were monitored for P. capsici infestation during the
growing seasons of 2002 through 2005. Site 1 is a creek which
flows into the river. Arrows indicate direction of water flow ....................... 48

Figure 3. Non-linear, quadratic regression curve of average water
Temperature (°C) during baiting periods from all sites and years
(2002 to 2005) and the percent incidence of P. capsici detection. ................ 55

Figure 4. Rainfall (weekly totals) and P. capsici detection data for
river monitoring sites in Allegan Co., Michigan in A) 2002, B) 2003,
C) 2004, and D) 2005. Gray vertical bars indicate rainfall (mm).
Black stars indicate positive P. capsici detection. ......................................... 58

Figure 5. Rainfall (weekly totals) and P. capsici detection data for
pond monitoring sites in Oceana Co., Michigan in A) 2002, B) 2003,
C) 2004, and D) 2005. Gray vertical bars indicate rainfall (mm).
Black stars indicate positive P. capsici detection. ......................................... 61

ix

Figure 6. Rainfall (weekly totals) and P. capsici detection data for
ditch monitoring sites in A) Lenawee (2004) and B) Oceana (2005)
Cos., Michigan. Gray vertical bars indicate rainfall (mm). Black
stars indicate positive P. capsici detection. .................................................. 62

Figure 7. Cluster analysis of P. capsici isolated from Michigan
surface water. Long term numbers (LT#) refer to culture numbers
maintained in the laboratory of Dr. Mary Hausbeck at Michigan
State University. Compatibility types (CT) are either A1 or A2.
Mefenoxam sensitivity (MS) characteristics are sensitive (S),
intermediately sensitive (IS), or insensitive (I). ............................................. 63

CHAPTER II

Figure 1. Field symptoms of P. capsici on bean. A,B) Snap bean
field from Cass Co., Michigan in August 2004. C, D) Yellow
wax bean field from Van Buren Co., Michigan in July 2005.
A-D) Note localized circular pattern of plant decline and death,
and association of plant decline with the surface water drainage
pattern. ........................................................................................................... 84

Figure 2. Symptoms of P. capsici on bean leaves, petioles, and stems.
A) Snap bean, ‘HyStyle,’ from Oceana Co., Michigan, 2003.
Note water-soaking on leaves. B) Snap bean, ‘HyStyle,’ leaf
from Cass Co., Michigan, 2004. Note water-soaked leaf
symptoms observed on leaf underside. C-G) Yellow wax bean,
‘Sunrae,’ from Van Buren Co., Michigan, 2005. C) Circular
necrotic lesion observed from leaf underside. D) Dark brown,
necrotic leaf and petiole lesions. E) Necrotic symptoms spreading
from junction of leaf and petiole. F) Brown, necrotic stem
symptoms. G) Close up of stem lesion with pathogen sporulation
on necrotic tissue ............................................................................................ 86

Figure 3. Symptoms of P. capsici on bean pods. A,B) Infected pods
of snap bean, ‘HyStyle,’ from Cass Co., 2004. A) Lesions on pods
are dark brown and sunken. B) Note brown, water-soaking symptoms
on pod tip where pod made contact with the soil. C,D) Infected
pods of yellow wax beans, ‘Sunrae,’ from Van Buren Co., Michigan,
2005. D) Close up of infected pod tip. Note water-soaking
and white pathogen sporulation on surface of necrotic pod tissue. ............... 87

Figure 4. Variation in cucumber fruit lesions (at 4 days post inoculation)
from inoculation with 6 select P. capsici isolates from Oceana Co.,
Michigan snap bean, 2003. A-C) Lesions from P. capsici isolates
from Field 1. Lesions vary from A) sunken and covered in dense
pathogen sporulation, which remains appressed to fruit surface, to
B) fluffy with dense aerial mycelium. C) Lesion of large diameter
with fluffy aerial mycelium. D-E) Lesions from P. capsici isolates
from Field 2. Lesions with D) moderate and E) dense aerial
mycelium. F) Lesion of largest diameter (when compared to A-E)
and moderate appressed pathogen sporulation. Numbers in the lower
right-hand corners of images indicate the isolate number. ............................ 91

Figure 5. Localized water-soaked foliar lesions on bean types caused
by P. capsici from Oceana Co., Michigan snap bean in 2003 (Field 1).
Lesions are depicted at 5 dpi under laboratory conditions. Symptoms
of localized water-soaking and necrosis are evident on A) ‘Bush
Tenderpod,’ B) soybean (leaf underside), C) ‘Bwush Lima,’ and D)
soybean (leaf surface). ...' ................................................................................ 93

Figure 6. Comparison of disease ratings (sporulation density,
sporulation diameter, and lesion diameter) for 6 select P. capsici
isolates from snap bean on cucumber fi'uit. Isolates 9951, 9948,
and 9974 are from Field 1 Oceana Co., Michigan, 2003. Isolates
9989, 9986, and 9988 are from Field 2 Oceana Co., Michigan, 2003.
Ratings were taken at 3 days post inoculation. Letters indicate
significant differences in disease among isolates using Tukey’s
Studentized Range Test at o.=0.05. ................................................................ 96

Figure 7. Average disease ratings of 6 P. capsici isolates fiom snap
bean on soybean leaves and petioles. Isolates 9951, 9948, and
9974 were from Field 1, Oceana Co., Michigan, 2003. Isolates
9989, 9986, and 9988 are from Field 2, Oceana Co., Michigan,
2003. Significant differences in petiole disease among isolates
are indicated by lower case letters within black vertical bars.
Significance was determined using Tukey’s Studentized Range
Test at a=0.05. No significant differences were found in leaf
disease among P. capsici isolates. Control plants exhibited no
disease. ........................................................................................................... 97

xi

Figure 8. Cluster analysis of P. capsici isolated from
Michigan snap bean. Long term numbers (LT#) refer to culture
identification numbers. Compatibility types (CTs) are either A1
or A2. Mefenoxam sensitivity (MS) characteristics are sensitive
(S), intermediately sensitive (IS), or insensitive (I). ...................................... 98

Figure 9. A-D) Segments of electropherograms from amplified
fragment length polymorphism (AF LP) profiles of genomic
DNA from four P. capsici isolates recovered from pumpkin
and snap bean in Michigan. The AF LP profiles were produced
using Beckman-Coulter CEQ capillary genetic analysis system
with the primer pair E-AC/M-CA and visualized using the CEQ
fragment analysis software. Polymorphic markers of 325, 362,
415, and 452 nucleotides are visible. ........................................................... 101

CHAPTER HI

Figure 1. Cucumber fruit production and inoculation technique.
A) Field production of cucumber fruit (10 days post planting)
at the Plant Pathology Farm, Michigan State University. B)
Inoculation chamber for screening cucumber fruit for resistance -
to P. capsici. C) Inoculated cucumber mu 3 days post inoculation
(dpi). D) Close up of inoculated cucumber hit 3 dpi (micro-
centrifuge tubes removed for disease evaluation). Note water-
soaking (ws) and pathogen sporulation (s). ................................................. 1 16

Figure 2. Comparison of disease ratings (sporulation density,
sporulation diameter, and lesion diameter) of 4 P. capsici isolates
(OP97, SP98, SFF3, SF3) on cucumber fruit during 1999 and
2000. No significant differences were found between isolates
using Fisher’s Protected LSD test (P=0.05). ............................................... 123

Figure 3. Fruit disease ratings of cucumber cultigens. A) Sporulation
density, B) sporulation diameter, and C) lesion diameter
measured from cucumber fruit inoculated with P. capsici
OP97. The number of cultigens are cumulative from 1999 to 2005.
Asterisks indicate where the cucumber standard ‘Vlaspik’ disease
ratings fell in comparison to other screened culti gens. Data from
2004 is omitted ............................................................................................. 124

xii

LITERATURE REVIEW

INTRODUCTION

Phytophthora is a major genus of plant pathogens within the Phylum Oomycota
which contains at least 60 species (14). The oomycetes appear to have closer
morphological affinities with algae and higher plants than with true fungi (44, 45).
Phytophthora blight, caused by Phytophthora capsici Leonian, is an important disease of
cucumber (Cucumis sativus L.), zucchini (Cucurbita pepo L. var. cylindrica Pans),
squash (Cucurbita pepo L. var. condensa Bailey), eggplant (Solanum melongena L.),
tomato (Lycopersicon esculentum Mill.), pepper (Capsicum annuum L.), pumpkin
(Cucurbita moschata), melon (Cucumis melon L.), watermelon (Citrullus lanatus L.), and
cacao (T heobroma cacao L.) (73). Phytophthora blight was first described on bell pepper
in New Mexico in 1922 (40). Since then, incidence of P. capsici on vegetable crops has
been reported in Colorado, Florida, California, Michigan, New York, Georgia, New
Jersey, North Carolina, and Europe (26, 66, 75, 76, 77, 120, 134, 135, 141, 143). The
incidence and severity of this disease has increased in recent years both within the US.
and worldwide (5). This pathogen is the limiting factor in the production of susceptible
vegetables despite the adherence to recommended control strategies (59). Crown, root,
and fruit rot caused by this soilbome pathogen has become a primary concern for
Michigan pickling cucumber growers (59, 78). Some producers have been forced to
abandon pickle production altogether as a result of increased crop loss from year to year
( 5 9). Spread of this disease threatens both fresh market and processing industries, and
limits the amount of production land for a large number of Solanaceous, Cucurbitaceous,

and more recently, Legumosaceous crops (29, 47, 100, 101).

Prolonged rain or irrigation leading to excessively wet soil conditions favors the
spread of the pathogen by splashing (125). Zoospores move in irrigation water, causing
spread within and among fields, potentially carrying inoculum from the epidemic site to
uninfested ground (59, 120). Narrow row spacing results in early closure of the leaf
canopy producing an ideal warm (>25°C), moist environment for disease progression
(106). Temperature and the availability of free water drive the initiation of early-season
disease and the ensuing production of secondary inoculum from infected tissue. Disease
symptoms on cucurbit plants include necrosis and wilting of the roots and crown.
Lesions on fruit first appear water-soaked, but quickly mature and take on a powdered
sugar appearance from accumulation of sporangia and/or oospores on fruit surfaces (5 9).
Fruit lesions develop beneath the leaf canopy and are the most frequently observed
symptom of P. capsici on cucumber. Infected fruit quickly break down both in the field

and in storage (59).

PATHOGEN LIFE CYCLE

The asexual phase of P. capsici includes the mycelial thallus, which produces
extracellular enzymes capable of macerating host tissue (37, 38). Mycelia differentiate
under ideal environmental conditions into long (35-138 mm) caducous pedicels which
give rise to semi- to fully-papillate limoniform sporangia (Fig. 1) (9, 37). At maturity,
deciduous sporangia break off and are dispersed by wind-driven rain or irrigation (38,
121). Sporangia are predominantly tapered at the basal end, can have up to three apices,
and are shaped variably depending on light, nutrients, and other environmental conditions

(3, 38). Sporangia] shapes include sub—spherical, ovoid, obovoid, ellipsoid, fusiform,

germinating oospore

0

,©\

© oospores
it“

direct

/""‘>

oospore germination

W

oogonia and antheridia

/ ) N
m '/ ’ 5/ ’, \‘
/ 3.) f ‘39
é)
Atty/\J T
b b
(b
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zoospores

6

direct sporangial germination

 

Figure 1. Disease cycle of P. capsici on pickling cucumber. Sexual (oospores) and

asexual (sporangia and zoospores) spores of P. capsici in association with a pickling

cucumber host. Oospores are produced when both A1 and A2 compatibility types come

into contact.

pyriform, and distorted. Under free water conditions, biflagellate (heterokont),
chemotactic, negatively geotropic zoospores are liberated from sporangia (Fig. l) (13, 37,
78, 84). An individual sporangium can release between 20 to 40 uninucleate zoospores,
which maintain motility in water for 5 to 10 hours (3 7). This increase in inoculum during
the early phase of an epidemic occurs rapidly, providing a competitive advantage in host
infection. Chlamydospores (hyphal survival structures) are not typically produced by P.
capsici isolates from pepper and cucurbit hosts. However, isolates from other hosts have
been reported to produce Chlamydospores under specific light and temperature conditions
(3 8).

Zoospores are predominantly responsible for the spread of the pathogen in water.
The negative geotaxis of zoospores in water (tendency to move toward the surface) may
be significant in their migration upward in a flooded sill (37). This migration
characteristic places them in an ideal position for dispersal by splashing in standing water
(3 7, 118). The attraction of zoospores to plant roots (chemotropism), as well as their
ability to stick to host surfaces during encystment, is advantageous to their function as
pathogens. The interaction of zoospores with host root exudates within the rhizosphere
has been well studied in a variety of host crops such as avocado and eucalyptus (51, 139,
149). More recently, molecules on the surface of P. capsici zoospores were shown to be
involved in reception of environmental signals that direct pre-infection behavior (12).
The root-zoospore interaction is responsible for much of the damping-off and seedling
disease associated with this pathogen.

As a heterothallic species, P. capsici oospores are produced sexually when

hyphae of A1 and A2 compatibility types (CT) interact (Fig. l) (108, 137). The

production of sexual structures (arnphigynous antheridia and spherical oogonia) is
induced hormonally in the presence of the opposite CT. Sexual reproduction is the major
source of genetic variation in nature (20, 66, 78, 80, 84, 87). Within Michigan and in
other US. regions, P. capsici exhibits an approximate ratio of 1:1, A1:A2 CT (59, 7 8,
79). An equal balance of the two CTs allows for frequent sexual recombination, and
thus, rapid development of resistance to chemical controls that target specific sites of
metabolic activity (80). In the US, P. capsici, P. cinnamomi, and P. infestans are the
only three documented heterothallic Phytophthora spp. which have both the A1 and the
A2 CT present to complete sexual reproduction (31, 33, 37, 38, 49, 149). However,
opportunities for sexual reproduction for P. infestans are limited because the CTs are
typically separated geographically (49).

Multiple factors affect oospore production. In most Phytophthora spp., oospore
production is optimal under darkness and temperature of less than 25°C (6, 52, 56, 72,
78, 99). Light becomes necessary for the germination of mature oospores. Yu et al.
(148) studied the effects of light and temperature on oospore production and maturation
of P. parasitica. Light had a significant impact on the induction of oospore production,
but was not necessary for maturation. Temperature affected the activity of the homione
needed to induce sexual reproduction. Other factors influencing oospore production in
many Phytophthora species include nitrogen, the atmospheric gas balance between 02
and C02, phospholipids, plant-derived compounds, vitamins, and sterols (37, 69, 97, 98).

The oospore structure from the central ooplast to the outer surface is comprised of

an inner oospore wall, outer wall, periplasm (in which nuclei and mitochondria are

oosphere

 

 

 

 

 

 

 

 

 

ooplast Inner oospore wall
, , outer oospore wall
lipids .
penplasm
oogonial wall
antheridial wall

 

 

Figure 2. Ultrastructure of a P. capsici oospore. The antheridial wall remains attached
to the oospore after fusion of the oogonia and antheridia. The average oospore diameter
is 20pm. Labeled oospore components include: lipids, oosphere, inner and outer oospore

walls, periplasm, oogonial wall, and antheridial wall.

sequestered), and oogonial wall (Fig. 2) (38). Oospores of P. capsici are predominantly
plerotic (they fill the oogonium), and have thick walls (2 to 6 um), which confer strong
resistance to external factors such as salinity and extreme temperatures. For this reason,
the oospores are the most durable and long-lived propagules in the soil (38). Oospores
can be formed not only in soil, but also on and within host tissue, wherever A1 and A2
CTs are present (2, 6, 34, 53, 63, 66, 73, 99, 122).

Oospores were hypothesized to be the main P. capsici survival propagules in soil
in New Jersey (13) and California (123); in Michigan, this hypothesis has been confirmed
(80). Oospores mature in 2 weeks to 3 months, depending on environmental conditions,
and remain viable for an extended period of time (5+ years). Germination occurs
predominantly by forming sporangia in water, root extract, and soil extract; however,
oospores can also germinate by forming direct-infecting germ tubes (63, 124). The
oospore germination rate in root and soil extracts is asynchronous and increases with time
of incubation (63). The ooplast (cortical layer in oospore) is consumed during
germination, with some of its lipid bodies moving into the cytoplasm of the emerging
germ tube. Most oospores produce a single germ tube that ruptures the thin outer oospore

wall and the oogonial wall near the oogonial stalk (37, 124).

PATHOGENICITY AND VIRULENCE

Phytophthora capsici inoculum (zoospores, sporangia, oospores, and mycelial
fragments) begins pathogenesis on a susceptible host with an initial chemical recognition
step that is often concurrent with the release of adhesive and degradative enzymes (37).
Yoshikawa et al. (146) observed that P. capsici produced a unique, active, extracellular
macerating factor when grown in culture. Propagules can directly penetrate the host plant
tissue with germination tubes. Germination tubes can grow into natural plant openings
such as stomata or wounds, or can differentiate to form appressoria which create a wound
in the plant surface by a penetration peg that uses hydrostatic pressure to puncture host
cells (11). Once inside host cells, P. capsici produces haustoria which gather plant
nutrients (60). After ramification through host tissue, the pathogen can proliferate by
producing sporangia and zoospores on host surfaces. Plants infected with P. capsici
exhibit altered metabolism and are often referred to as ‘sinks’ because the reduction in
photosynthetic potential causes the plant to send resources to sites of infection (3 7).
Infection is chronic and results in plant death. Oospores are often produced on necrotic
host tissue and are released into soil afier tissue decomposition.

Solanaceous, Cucurbitaceous, and recently Leguminosaceous plant families were
all found to be susceptible to P. capsici infection (47, 59). Among these families there
are differences in susceptibility based on plant variety, age, and organ (3 7). Developing
fruit of a pickling cucumber plant are relatively more susceptible than the vine, crown,
and roots (59). However, fully mature fruit exhibit some resistance to infection (1).

Compared to other host plants, yellow summer squash exhibit elevated susceptibility and

rapidly succumb to disease (59). Bean roots remain free of disease from P. capsici, yet
all above-ground parts are susceptible to infection (47). While these differences
represent phenotypic variations of resistance in the host, there are also variations in
virulence of the P. capsici isolates (47, 75, 86, 117, 132).

Polach and Webster (117) suggested the existence of physiological races of P.
capsici based on the response of different host genotypes (differentials) to pathogen
isolates. Strains were identified by a series of inoculations with 23 isolates on 5 different
susceptible plants from the Solanaceous and Cucurbitaceous families. Among the
isolates tested, fourteen were distinguished by their selective pathogenicity (1 l7).
Oelke, et al. (109) also suggested the presence of physiological races of P. capsici as the
cause of breeding limitations in pepper. The disease response of pepper varieties
inoculated with isolates of P. capsici indicated that some isolates were responsible for
root rot, whereas other isolates only caused foliar blighting. The isolates were described
as unique races based on pathogenicity (109).

Although diversity in virulence among P. capsici isolates from multiple hosts has
been noted, it is questionable whether this diversity reflects true host specificity (68, 75,
86, 117). Studies on resistance of pepper and tomato cultivars to different isolates of P.
capsici indicate that expression of host resistance is affected primarily by environmental
factors such as inoculum dose, temperature, soil moisture, and plant age (66, 75, 111,
119). In the pumpkin- and pepper-P. capsici pathosystems, there are putative cases of
specific host-parasite interactions or specializations. However, absence of interaction

seems to be the rule (86, 111). In general, distinct host specificity among races has not

10

been confirmed; and host genetics conveying limited quantitative resistance are complex
(37,124)

The host range of P. capsici was evaluated extensively by Satour and Butler in
1967 (123). Disease symptoms were observed on 45 species of cultivated plants and
weeds, representing 14 families of flowering plants (123). Tian and Babadoost also
reported P. capsici infection on beet, Swiss chard, turnip, spinach, and lima bean (132).
Field reports of P. capsici causing disease on bean cultivars have recently been made
from Delaware, Maryland, New Jersey, and Michigan, with select Michigan P. capsici
isolates also causing symptoms on soybean under laboratory conditions (29, 47, 100,

101).

BAITIN G FOR PH Y T OPH T H ORA SPP.

Methods used to detect, isolate, and quantify Phytophthora spp. in soil are
numerous, but their success varies depending on the species (37, 38, 84, 96). The most
common method is soil dilution plating using a selective medium such as BARP
(benomyl-, ampicillin-, rifampicin-, and pentachloronitrobenzene-amended, unclarified
V-8 juice agar). Homer and Wilcox (64) optimized a technique called Soil Air Dried
And Moistened Chilled And Plated, SADAMCAP, which utilizes basic culturing
protocols to increase the number of P. cactorum colonies on agar media. This method
allowed an accurate and reproducible quantification of P. cactorum colony-forming units

derived from apple orchard soils of New York. Although this direct soil to agar plating

ll

technique was successful for P. cactorum, a similar method had limited success for P.
capsici (64, 65).

Papavizas, et al. (114) developed a selective medium that inhibited most soil
micro-organisms except for oomycetes. This medium was used to develop a dilution
plating technique for the direct isolation and quantification of P. capsici from soil and
infected pepper plants. The isolation technique from peppers was successful. However,
in the case of soil, more than 46 propagules per gram of soil needed to be present for P.
capsici to be detected, while naturally infested soil averaged only 0-24 propagules per
gram (1 14).

Oospores present in soil have been the most difficult propagules to detect and
quantify because they do not germinate readily in culture and generally are not accounted
for in soil assays (37, 84, 116). In addition, Phytophthora oospores are morphologically
similar to those of Pythium, which are ubiquitous and numerous in agricultural soils.
Thus, baiting with susceptible seedlings or fi'uit remains the most successful tool for the
detection of Phytophthora spp. in soil (119, 138).

To detect P. capsici from soil using a bait method, a susceptible bait from a field
without a history of P. capsici infestation is needed. With an infected seedling or fruit,
the presence or absence of P. capsici in a given soil sample can be determined and
isolates acquired for further investigation. Some baiting assays detect a lower level of
inoculum than may actually be present (37, 38, 84, 96). However, other baiting
techniques are more sensitive than selective media methods, and have the capability of

detecting just one propagule of Phytophthora per gram of soil (125).

12

Water baiting techniques have been successful for detecting P. cactorum and
other species in infested irrigation water and for timing application of chemicals for
disease control (112, 116, 145). In Washington State (145), green pears were used to
bait Phytophthora spp. pathogenic to tree fruit. Washington fruit growers regularly begin
treating their irrigation water with copper when immature pears, which are suspended in
irrigation canals, become infected with P. cactorum, the causal agent of sprinkler rot.
Infected pear baits were surface sterilized and diseased tissue was excised from lesion
margins. Tissue was incubated on selective medium at room temperature. Positively
identified P. cactorum cultures were then further analyzed (145).

In a study of Phytophthora spp. associated with cranberry root rot in surface
irrigation water in New Jersey, a lupine baiting technique was utilized in streams,
irrigation reservoirs, and drainage canals (112). A 10- to 15-day-old lupine seedling was
secured in a Styrofoam boat with its roots extending into the water source. The seedling
was then removed 48 hours later and the roots were cut from the seedling. The roots
were rinsed and plated onto a selective medium and observed 2 to 4 days later. Positive
identification of both P. cinnamomi and P. megasperma from critical water sources was
achieved using this technique (112, 116).

Tomato leaf disks and green tomato fruit were used as baits in a study designed to
monitor the spread of P. parasitica (causal agent of buckeye rot) in irrigation furrows
(107). Baits were floated on the surface of water that was experimentally infested with P.
parasitica. Both baits were useful for detecting the pathogen at various distances from
inoculation source points. In this same study, detection of P. parasitica was also carried

out by dilution plating but the recovery rate was low (107). Pepper leaf disks have also

13

been used in assaying flooded soil samples for P. capsici presence (83). In these assays,
containers of field soil were flooded with water, and the disks were floated on the surface
and analyzed 72 hours later. Phytophthora capsici successfully colonized leaf disks in
the containers with infested soil (83).

Recovery of Phytophthora spp. from water has also been achieved without the use
of baits. A filter-based method using hiydrophilic membranes was carried out with
recycled nursery irrigation water (37). Organisms deposited on filters were plated for
identification and quantification (61). This technique was used to monitor levels of
Phytophthora and Pythium spp. present in the components of a recycling irrigation
system at a perennial container nursery in Virginia over a two-year period (17). The
relative occurrence of Phytophthora and Pythium spp. as well as their taxonomic
diversity were docmnented for three different water sources. To validate the filter-based
method, rhododendron leaf disk baits were also implemented (1 7). Baits were surface
washed and placed onto selective media for oomycetes. Phytophthora and Pythium spp.
were identified morphologically and successfully quantified. Results of rhododendron

leaf baits confirmed the efficacy of the filter method (17).

MANAGEMENT

Knowledge of field disease, crop, and chemical history is a necessity in
controlling P. capsici effectively (59). Some crops lend themselves to cultural practices,
such as constructing raised beds for hand-picked zucchini. However, control measures

need to be heightened for a crop such as pickling cucumbers that is mechanically

14

harvested. Multi-faceted disease-management practices, combining cultural and
chemical control in addition to genetic host resistance are necessary (5 9).
Cultural Management

Cultural techniques that have aided in limiting P. capsici spread and infection
have included row spacing, crop rotation, water management, bedding and plasticulture,
and avoidance of low-lying or infested fields (18, 59, 66, 127). In Michigan, row spacing
for machine-harvested pickling cucumbers varies from 11 to 30 inches with a plant
population of 35,000 to over 100,000 plants per acre (103, 105). A closed plant canopy
promotes high relative humidity and sustained soil moisture after a rain or irrigation
event (105). Under these conditions P. capsici rapidly develops sporangia and
subsequently releases a large number of zoospores. A 2003 study by Ngouajio ( 102, 104)
examined the effects of widened rows on canopy dynamics and fruit yield. Cucumber
canopy remained open during most of the growing season when wide rows (61.0 and 76.2
cm) were used. The optimum row spacing for maximizing the number of fi'uits per plant
was 61.0 cm. With 76.2-cm rows, yield was only slightly reduced. These results suggest
that it is possible to significantly reduce cucumber plant density without reducing yield
(104,106)

A two-year rotation with a non-host crop has been suggested to avoid buildup of
P. capsici; however, planting any host crops into a field with a history of P. capsici is
risky since non—host rotations do not eliminate the pathogen (59). Lamour and Hausbeck
investigated the survival of P. capsici in the field following a typical 2-year crop rotation
(2 years of production with crops not susceptible to P. capsici) in Michigan (81). The

potential for dormant inoculum (oospores) to survive a 30-month non-host period was

15

confirmed, with molecular tools, indicating that management strategies relying on crop
rotation may not provide economic control of P. capsici (81).

Managing field water includes using fields with soils that allow for efficient
drainage, avoiding low-lying areas where water may stand, and limiting irrigation (18,
59). Furthermore, it is recommended that irrigation on pickling cucumbers within a week
of harvest be limited for maintaining healthy fruit at a critical, susceptible stage (5 7, 59).
Sources of irrigation water that receive run-off from fields with a history of P. capsici
should also be avoided (46). Water management remains critical post-harvest in
transportation wagons and bulk bin storage since susceptible fruit can still become
infected even after removal from the field (57, 59).

When harvest methods allow, planting host crops on raised beds (6 inches in
height minimum) greatly reduces the risk and incidence of P. capsici infection (5 9). To
complement the beneficial effects of raised beds, black plastic application over beds with
the addition of sub-plastic drip-tape irrigation further enhances successful crop
production in an infested field (59, 126). These methods facilitate tight control of soil
moisture and reduce splashing of water from contaminated soil onto plant parts. In
addition, susceptible fruit have increased protection from direct contact with soil, where
pathogen propagules reside (59).

Chemical Management

There are few chemical products available that provide economically acceptable
control of P. capsici (59). Since 1977, metalaxyl has been used to control plant diseases
caused by oomycetes (15). Metalaxyl (Ridomil) is a systemic phenylamide fungicide

with a site—specific mode of action that inhibits RNA polymerase activity and can be

16

applied by drench, seed treatment, and by foliar application (15, 24). Mefenoxam
(Ridomil Gold) is the active isomeric form of metalaxyl, which comprises 50% of
metalaxyl, whereas mefenoxam consists of 100% active isomer (115). Metalaxyl is
toxic to sporangia and zoospores in vitro, yet has little effect on sporangium and zoospore
germination (28). It delays expansion of young lesions and suppresses sporangium
production on treated plants (15, 24, 87). Resistance to metalaxyl was first reported in P.
Ainfestans in Ireland in the late 19705, in the Netherlands in 1981, and has since been
reported frequently in both homo- and heterothallic Phytophthora species (27, 32, 41, 49,
82, 87, 133). In P. infestans, resistance occurs as a result of a simple change in the target
site and there is no correlation between resistance and other phenotypes, such as CT or
virulence (30, 48). Lamour and Hausbeck (80) found that in vitro, mefenoxam sensitivity
in P. capsici is conferred by a single, incompletely dominant gene.

When fungicide selection pressure is removed, reversion to mefenoxam
sensitivity within a P. capsici population does not occur within an agriculturally
significant time period (2 years) (80). Application of mefenoxam to a field with a
significantly insensitive P. capsici population has no management benefit and can
increase economic loss to a grower (82, 115). Pathogen insensitivity to mefenoxam has
limited the usefulness of this fungicide worldwide (43, 70, 71, 78, 80, 87). Few
alternative products are currently available that match the effectiveness that mefenoxam
provides against a sensitive pathogen population (59).

Released in 1988, dimethomorph (DMM) (Acrobat), a derivative of cinnamic
acid, is a systemic fungicide with supposed protectant, curative, and antisporulant

activities against members of the Peronosporaceae and the genus Phytophthora (19, 37).

17

DMM interferes with the assembly of wall polymers in the fungal cell (23). As a result,
zoospores fail to produce cell walls, and hyphae stop expanding. There is no cross-
resistance between DMM and phenylamide fungicides, such as mefenoxam.
Phytophthora infestans was recently reported to have little or no risk of developing
resistance to DMM in nature (129) and there are no reports of resistance in P. capsici.
This characteristic may ensure prolonged efficacy of post-infection applications (129). In
addition, the fungicide is highly effective at relatively low rates (23). Only DMM was
effective in managing disease once mefenoxam insensitivity had been established (5).
Further studies by Hausbeck et al. (59) on pickling cucumbers, also support this
conclusion.

Zoxamide (Zoxium), a non-systemic benzamide, has been recently introduced as
an oomycete fungicide that acts on pathogen microtubules (147). Although field studies
suggest that the risk of the development of insensitivity in the pathogen population is
much lower in the field for zoxamide than for mefenoxam, it is a site-specific fungicide,
and precautions must be taken with its use (147). Zoxamide application, in combination
with copper hydroxide (Kocide), provided effective control of P. capsici zoospore
infection on detached cucumber fruit (Harlan, BR, and Hausbeck, M.K., unpublished
data). However, trials with this product showed a reduced level of protection when
compared to DMM (4, 67).

Alternative compounds have also been tested for their Phytophthora disease
management potential. Simple additions of compounds such as gypsum to field soil was
shown to limit zoospore production of P. cinnamomi (94). Sodium tetrathiocarbonate

(STTC) and water applied to soil was reported to have some fungicidal effects on

18

Phytophthora spp. in greenhouse trials at high rates of application (90). Soils amended
with myxobacteria have been demonstrated to reduce grth of P. capsici by enzymatic
inhibition (16). The roles of calcium, phosphite, surfactants, and inducers of host plant
resistance (chemical and non-pathogen) have also been investigated for their disease
management properties (21, 22, 25, 36, 39, 42, 54, 55, 74, 85, 93, 95, 110, 127, 128, 140,
144). Although alternative compounds may provide some management benefit, the
methods and rates of application of such products must be consistent, effective, and
economical for large scale production.

The multiple propagule types of P. capsici are each affected differently by
fungicides. In an in vitro study, Matheron and Porchas (91), observed the impact of five
fungicides on the growth, sporulation, and zoospore cyst germination of P. capsici. In
addition, effects of these chemicals on the development of root, crown, and fruit rot of
chile pepper were studied (91, 92). Inhibition of mycelial growth, sporangial formation,
and cyst germination was greatest when the pathogen was treated with DMM. However,
DMM had little effect on the inhibition of zoospore motility. Fluazinam had a minimally
inhibitory effect on mycelial grth and cyst germination. However, this product
inhibited zoospore motility by almost 100% at all concentrations and was equally as
effective as DMM in inhibiting sporangial formation. Azoxystrobin had a moderately
inhibitory effect on mycelial growth and was most effective at higher concentrations on
sporangial formation, zoospore motility and cyst germination. Metalaxyl had a
significant limiting effect on the growth of myceliurn, sporangial formation, zoospore
motility, and cyst formation. The most effective compounds on pepper stems and fruit

were mefenoxam and DMM (91).

19

Genetic Host Resistance

Breeding efforts have resulted in two pumpkin cultivars exhibiting quantitative
resistance due to thick rinds: ‘Danmatmaetdol’ from the National Institute of Agricultural
Science and Technology at Suwon, Korea (86) and ‘Lil Ironsides’ from Harris Moran
Seed Company (Modesto, CA). Increased cuticular thickness has also been implicated in
elevated disease resistance to P. capsici in Mexican-type pepper fruit (10). No pepper
cultivars have been shown to have universal resistance to P. capsici root and foliar
blighting (7, 109), although ‘Paladin’, a commercially marketed pepper hybrid from
Rogers/Novartis is marketed as P. capsici tolerant.

Traditional cucurbit breeding for beneficial horticultural traits involves simple
cross-pollination of parents with desirable qualities. Compatible cross-pollination, or
intercrossibility, can occur only with hard squash, pumpkins, and gourds. Melons,
summer squash, and cucumbers are not intercrossible; therefore, resistance present in
melons cannot be bred into cucumbers by traditional methods (142).

Pepper plants produce the phytoalexin capsidiol and can upregulate pathogenesis-
related (PR) genes known to encode for multiple plant defense proteins following
infection (35, 37, 88, 89, 109). Recent research on tomato showed expression of PR-l
proteins when challenged by P. capsici (61). These findings provide evidence of plant
response systems, which can be exploited for potential use in a breeding program. An
example of this application is a marker-assisted selection program, in which four
quantitative trait loci (QTL) for P. capsici resistance in a small-fruited pepper line were
successfully introduced into a susceptible bell pepper line (130, 131). The selection

program was initiated from a doubled-haploid line issued from the mapping population

20

and involved three cycles of marker-assisted backcrossing. Resulting bell pepper plants
expressed quantitative resistance to P. capsici (131).

Selection of appropriate inoculum and inoculation techniques is critical when
screening for disease resistance (113, 136). The rhizosphere of host plants prone to root
rots is an important feature to consider. In France, pepper breeders have set up an
inoculation method involving incubation of mycelium in the proximity of plant roots in a
liquid medium to best mimic the rhizosphere of pepper in a disease situation. Their
experimental methods have made it possible to breed for constitutive as well as induced
resistance components in controlled conditions at the seedling stage (113). Although
inheritance of host resistance to P. capsici is understood to be controlled by two distinct
genes in pepper, there is little information regarding the inheritance of resistance to P.
capsici in other host plants (86, 117).

To date, much work has been carried out in effort to curb P. capsici. However, no
single management practice provides complete control in a reliable and sustainable
manner. Crop rotation, sanitation, management of field moisture, and judicious use of
fungicides are relied upon to maintain profitability of vine crop production. Although
rotation to supposed non-susceptible hosts has been studied for control against this
pathogen, it does not provide effective control since the pathogen survives in soil for a
long time (>5 years anecdotal evidence, M.K. Hausbeck), and the pathogen has now been
isolated from a common rotational crop, snap bean (58). Field moisture, in areas of high
relative humidity or rainfall, cannot be effectively managed and is therefore not a stand-
alone control option. Because completely resistant cultivars have not been found,

research efforts toward control have been focused on the biology of the pathogen. The

21

research objectives of this dissertation include investigating irrigation water as a means of
zoospore spread, documenting and characterizing P. capsici on snap bean, and screening

cucumber germplasm for fruit resistance to P. capsici.

22

10.

ll.

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35

CHAPTER I

BAITIN G PH Y T OPH T HORA CAPSICI FROM MICHIGAN SURFACE

IRRIGATION WATER AND CHARACTERIZATION OF ISOLATES

36

ABSTRACT

The fungal-like oomycete Phytophthora capsici causes disease on cucurbit and
solanaceous crops worldwide. In free water, P. capsici sporangia release zoospores
which may be transported by moving surface water. Surface irrigation sources (river
system, ponds, and ditches) in three Michigan counties with history of susceptible crop
production were monitored for P. capsici during four growing seasons (2002 to 2005).
Pear and cucumber baits were suspended in water at monitoring sites for 3 to 7 days and
water temperature was recorded. Baits were washed and lesions excised and cultured on
amended water agar. Phytophthora capsici was detected at monitoring sites in multiple
years despite nearby crop rotation to non-hosts. Recovered isolates (270) were screened
for sensitivity to the fungicide mefenoxam and characterized for compatibility type (CT).
Mefenoxam insensitivity of isolates was common in water sources from southwest and
southeast Michigan. Most monitoring sites yielded isolates of a 1 :1 ratio of A1 :A2 C Ts.
AFLP analysis of select isolates from 2002 to 2004 indicated a lack of similarity groups
persisting over time and in specific geographical locations. Phytophthora capsici was not
found to overwinter in the eleven Michigan surface water sources monitored. The
consistent detection of P. capsici in surface water used for irrigation in Michigan

suggests that this is an important means of pathogen dissemination.

37

INTRODUCTION

Phytophthora capsici [Leonian] infects Solanaceous and Cucurbitaceous hosts
including cucumber, eggplant, tomato, pepper, pumpkin, squash, melon, and zucchini (7).
Recently, snap beans have been added to this list of susceptible crops (4). In addition to
crown rot, P. capsici causes fi'uit rot symptoms which appear initially as water-soaked
lesions that expand rapidly. Sporangia and/or oospores develop in the lesions resulting in
fruit surfaces with a powdered sugar appearance. Infected fruit quickly break down both
in the field and post-harvest (7).

Oospores overwinter in soil and plant debris and initiate primary plant infections
in the spring (1 l, 12, 14). Oospores are produced when mycelium of A1 and A2
compatibility types (CTs) come into contact (18, 21). Both CTs have been documented
in naturally-infested fields in Michigan (11, 12, 13) and other states (7). Mycelia and
sporangia develop on infected host tissue under warm (ZS-28°C), wet conditions.
Sporangia and zoospores are secondary asexual inoculum produced during the growing
season, which cause rapid escalation of disease (2, 3). In free water, lemon-shaped
sporangia release 20-40 motile zoospores, each capable of causing infection (7).

Phytophthora capsici management requires a multi-faceted approach of both
cultural and chemical strategies. Planting P. capsici-susceptible crops in well-drained
fields, on raised beds, and avoiding low—lying regions help to manage disease.
Conservative irrigation, especially close to harvest is recommended (7, 20). When
applied preventively and frequently, fungicides can also limit disease (7). However,
resistance of P. capsici to the historically-used fungicide mefenoxam has been widely

documented in Michigan and other vegetable production regions (12, 13). Because

38

current measures, even when used in a comprehensive program are often not adequate,
preventing the introduction of P. capsici into new sites is critical (7).

Oomycetes have been identified in surface water used in various commercial
plant production systems (1, 8, 15, 17, 19, 25). Phytophthora and Pythium spp. have
been detected in recycled water used for irrigation in greenhouse and nursery production
in Virginia (1, 8). Phytophthora cactorum was identified in surface water used for
irrigation of pear in the state of Washington (25). Both P. cinnamomi and P.
megasperma were detected in surface water used in a cranberry production system in
New Jersey (19). In North Carolina (15) and California (17), P. parasitica was detected
in furrow irrigation water which moved the pathogen from loci of infestation to parts of
the field that were uninfested. Phytophthora capsici has not yet been documented in
surface water and many Michigan vegetable growers rely on surface water for irrigation
of susceptible crops.

My objective was to determine whether P. capsici is present in surface water used
for irrigation in Michigan and characterize the CT and sensitivity to mefenoxam of the
collected isolates. Representative isolates were subjected to amplified fragment length
polymorphism (AF LP) analysis to determine if genetic similarity groups are present over
time and among geographical locations. Water and rainfall were also monitored and

analyzed for correlation to P. capsici incidence.

39

MATERIALS AND METHODS

Site Selection

Water monitoring sites were either adjacent to a vegetable production field with a
history of P. capsici infestation or adjacent to a field with a crop infected with P. capsici
during the 2002 to 2005 growing seasons. Sites were selected in three of the primary
vegetable production regions of Michigan: northwest (NW; Oceana Co.), southwest
(SW; Allegan Co.), and southeast (SE; Lenawee Co.). Baiting was usually initiated in the
spring and terminated at the end of the growing season (Tables 1-3).
Baiting, P. capsici Identification, Water Temperature, and Rainfall

Baiting traps were constructed by attaching Styrofoam pool noodles to plastic
milk crates with a securable lid (Fig. 1A). Each trap contained two green pears,
purchased from a local supplier, and a cucumber produced at the Michigan State
University Plant Pathology farm, a site without a history of P. capsici. Pears were
selected as baits for P. capsici because they had previously been shown to be susceptible
to Phytophthora spp. when used in a water system (25). A WatchDog data logger
temperature sensor from Spectrum Technologies, Inc. was attached to the milk crate to
measure and record water temperature each hour (Fig. 1A). Each trap was secured to a
fixed point and deployed into one of the following water sources: river, creek, pond, or
ditch (Fig. 1B). Baits and temperature sensors were replaced once or twice weekly.
Hereafter, a baiting period refers to the time that the baits remain in the water (3-7 days).
After removal from the traps, cucumbers and pears (Fig. 1C and D) were rinsed in
distilled, de-ionized water, dried, and tissue from water-soaked lesions was placed onto

water agar amended with rifampicin (2 m/L) and ampicillin (2 ml/L). After 5 days of

40

Table 1. Time of sampling and P. capsici detection for SW Michigan (Allegan County)

 

 

 

 

 

 

 

river sites, 2002 to 2005.
Site:Type Year Initiation Date of First # of periods Crop in nearby
Date Detection with field
P. capsicia

1: Creek 2002 18 J unb 7 Aug 11 cucumber"
2003 30 Mayc 25 Jul 10 potato
2004 12 Mayd 27 Aug 2 soybean
2005 3 May" 26 Jul 5 com

2: River 2002 18 Junb 19 Aug 3 com
2003 22 MayC 22 Aug 1 cucumberr

3: River 2002 18 Junb 19 Aug 3 cucumber'
2003 22 Mayc 18 Aug 1 corn

4: River 2003 30 Mayc 11 Aug 2 cucumber'
2004 12 Mayd 20 Jul 6 cucumberf
2005 3 May" 26 Jul 2 cucumberf

5: River 2003 30 Mayc 23 Sep 1 cucumber’
2004 12 Mayd 3 Aug 2 cucumber"
2005 3 Maye 29 Jul 1 soybean

6: River 2003 30 Mayc 1 Aug 2 cucumberr
2004 12 Mayd 17 Aug 2 cucumber"
2005 10 Maye 9 Aug 2 potato

 

2'Baiting period was 3-7 days during which baits were in surface irrigation water.
bIndicates baiting termination occurred on 8 October 2002.

cIndicates baiting termination occurred on 28 October 2003.

dIndicates baiting termination occurred on 22 October 2004.

°Indicates baiting termination occurred on 21 October 2005.

rIndicates that nearby crop exhibited disease caused by P. capsici.

41

Table 2. Time of sampling and P. capsici detection for NW Michigan (Oceana Co.)

 

 

 

 

pond sites, 2002 to 2005.
Site:Type Year Initiation Date of First # of periods Crop in
Date Detection with nearby field
P .capsici'
1: Naturally- 2002 20 Augb 20 Aug 8 yellow squasb’
fed

2003 16 AprC 22 Aug 2 snap beant
2004 20 Aprd 31 Aug 1 carrot
2005 19 Apr" 3 Aug 2 corn

2: Well-fed 2002 18 Junb N/A 0 snap bean
2003 16 Julc N/A 0 yellow squash"

3: Well-fed 2002 25 Junb 20 Aug 1 carrot

 

aBaiting period was 3-7 days during which baits were in surface irrigation water.
bIndicates baiting termination occurred on 10 October 2002.
cIndicates baiting termination occurred on 28 October 2003.
dIndicates baiting termination occurred on 23 October 2004.
°Indicates baiting termination occurred on 21 October 2005.

fIndicates that nearby crop exhibited disease caused by P. capsici.

42

Table 3. Time of sampling and P. capsici detection for Michigan ditch sites (Lenawee

and Oceana Cos.), 2004 to 2005.

 

 

 

Site:Type Year Initiation Date of First # of periods Crop in
Date Detection with nearby field
P. capsici‘l
1: Run-off 2004 20 Julb 5 Aug 5 cucumberd
SE Michigan '
(Lenawee Co.)
2: Culvert 2005 27 Julc 29 Jul 7 zucchini“
NW Michigan
(Oceana Co.)

 

aBaiting period was 3-7 days during which baits were in surface irrigation water.
bIndicates baiting termination occurred on 21 October 2004.

cIndicates baiting termination occurred on 7 October 2005.

dIndicates that nearby crop exhibited disease caused by P. capsici.

43

5“.” .. r ’
6:139 0 'r
‘3

‘ .;¥'..u.» g
u» '1 1', 4

 

Figure 1. A) Baiting station for P. capsici and contents: two green pears,
one cucumber from an uninfested source, and a temperature sensor. B)
Baiting station deployed in a pond used for crop inigation. C) Cucumber
bait retrieved after one week in water monitoring site. D) Pear bait retrieved
afier one week in water monitoring site. (Images in this dissertation are

presented in color).

incubation, plates were observed with a compound microscope and P. capsici was
identified based on morphological characteristics according to the Phytophthora spp. key
by Waterhouse, 1963 (24). Data from temperature sensors were downloaded using Spec
Ware 6 data logger software (Spectrum Technologies, Inc., Plainfield, IL) and hourly
data were averaged for each baiting period. Rainfall data for each baiting location were
collected from nearby weather monitoring stations, totaled for each baiting period, and
were analyzed using nonlinear polynomial regression analysis.
Compatibility Type Determination

Compatibility types of P. capsici isolates were determined by placing agar plugs
(7.0-mm diameter) from the edge of expanding single-zoospore-derived cultures onto a
V-8 juice agar plate. At a distance of 3 cm from the baiting isolate plug, a 7.0-mm plug
of a known A1 or A2 CT was placed. Known standard isolates of P. capsici were OP97
(A1 CT) isolated from pickling cucumber fruit in NW Michigan in 1997; and SP98 (A2
CT) isolated from pumpkin fruit in SW Michigan in 1998. Culture numbers refer to
isolates maintained in the Phytophthora stock collection in Dr. Mary Hausbeck’s
laboratory in the Department of Plant Pathology, Michigan State University. After 7 to
10 days incubation at 23-25°C under darkness, plates were observed for the presence of
oospores between the two plugs.
Mefenoxam Sensitivity Determination

Mefenoxam sensitivity was determined by placing a 7.0-mm agar plug from a
single-zoospore-derived culture on V-8 juice agar and V-8 juice agar amended with 100
ppm mefenoxam (Ridomil Gold EC, 48% a.i. suspended in sterile distilled water and

added to cooled agar). Plates were incubated at 23-25°C for 3 days, and colony

45

diameters were measured. Growth of an isolate on amended media as compared to
unamended media was classified as sensitive (S, <30% of the control), intermediately
sensitive (IS, 30-90% of the control), and insensitive (1, >90% of the control) (10).
DNA Extraction and Amplified Fragment Length Polymorphism Analysis

Genomic DNA was extracted from approximately 10 mg of freeze-dried P.
capsici mycelium using DNeasy® mini-kits (Qiagen, Valencia, CA., USA). Mycelium
was grown in antibiotic-amended V8 broth. DNA was quantified on an agarose gel with
known standards. EcoRI was obtained from Invitrogen (Carlsbad, CA., USA), while
MseI and T4 DNA ligase were obtained from Takara (Madison, WI., USA). EcoRI and
MseI adapters and primers for ligation and amplification reactions were obtained from
Integrated DNA Technologies (Coralville, IA, USA). For sequences of the adapters and
primers, see Vos et al. (23). The fluorescently labeled primers were obtained from
Proligo (Boulder, CO, USA) and are comprised of the EcoRI core sequence with and
without any selective nucleotides and either the WellRED D4-PA label (Proligo) or the
WellRED D3-PA label at the 5’ end.

Restriction, ligation, preamplification, and selective amplification reactions were
carried out as described by Habera et al. (6). Fluorescent products from the selective
amplifications were analyzed on a CEQTM 8000 Genetic Analysis System (Beckman

Coulter, Fullerton, CA, USA) using the manufacturer’s protocols.

46

RESULTS

Pear and cucumber fruit were suitable baits for detecting P. capsici in Michigan
surface water. Cucumber fruit were especially susceptible and sensitive as baits, with
approximately 70% of all P. capsici isolates collected from symptomatic cucumber. In
addition to P. capsici, other oomycete organisms were isolated from baits. Based on
morphological characteristics and AF LP analysis, Pythium spp. were identified each year.
Pythium spp. were prevalent during the early (April and May) and late (September and
October) portions of the growing season. Additional Phytophthora spp. were also
observed during the four years of water monitoring.
River System

Prior to 2002, pickling cucumbers were routinely planted in fields nearby the SW
Michigan (Allegan Co.) river system (Fig. 2). One or two years of non-host rotations to
corn and soybeans were common. Phytophthora capsici was detected in the six river
sites monitored during the 4-year period. While baiting was initiated in the spring and
early summer (prior to 18 June), P. capsici was never detected prior to 20 July. The
pathogen was detected at each site for multiple years even when a P. capsici-host crop
was absent in nearby fields. In creek site 1 (Fig. 2), ll baiting periods (from 7 August to
1 October) during 2002 yielded P. capsici. Detection of the pathogen continued for an
additional three years despite a nearby crop rotation of potato, soybean, and corn (Table
1). Due to the high number of baiting periods (10) with positive P. capsici identification
at creek site 1 during 2003, potato plants in the nearby field were sampled, but the

pathogen was not detected. At river sites 2-6, when a nearby field was planted to

47

 

. *
S"? 1 Site 4
Site 6 a Site 5

. *
5““ 3 * Site 2

1 mile

 

 

 

 

Figure 2. Schematic of six sites along a river and creek system of SW Michigan which
were monitored for P. capsici infestation during the growing seasons of 2002 through
2005. Site 1 is a creek which flows into the river. Arrows indicate direction of water

flow.

48

cucumber, P. capsici disease symptoms were observed and included fi'uit with water-
soaked lesions and pathogen sporulation (Table 1).
Ponds

Phytophthora capsici was detected in a naturally-fed pond (Site 1) and a pond fed
by a deep well (Site 2). In a second well-fed pond (Site 3) with nearby field Sites
historically infested with P. capsici, the pathogen was not detected. Baiting in the
naturally-fed pond (Site 1) was initiated late in the 2002 growing season in response to
crop loss in an adjacent field from P. capsici. The pathogen was detected immediately
after bait initiation and throughout the remainder of the growing season. Yellow squash
was planted in the field nearby the monitoring site in 2002, snap bean in 2003, and non-
host crops were planted in the field for the remaining 2 years of the study. Phytophthora
capsici was isolated from infected yellow squash plants in 2002 and from snap beans in
2003 (Table 2). No P. capsici was recovered from sampled carrots in 2004 (Table 2).
Well-fed pond site 3 had just a single baiting period with positive P. capsici detection. It
is likely that this infestation occurred as a result of an influx of run-off water from a
neighboring field with diseased zucchini following an irrigation event.
Ditches

Limited data were collected from ditch sites used for irrigation. In 2004,
monitoring was initiated in response to widespread and severe disease in a hand-
harvested cucumber field in SE Michigan (Lenawee Co.). The detection of P. capsici at
this site represents the first confirmation of this pathogen in water used for irrigation in
SE Michigan. Despite our interest in continuing to monitor this site in 2005, the grower

declined. The 2005 ditch Site represented a culvert that received run-off from a number

49

of fields, some with a known history of P. capsici infestation. The pathogen was detected
immediately following baiting initiation and throughout the remainder of the growing
season (Table 3).
Compatibility Types

For all monitoring Sites where more than one P. capsici isolate was obtained, both
Al and A2 CTS were typically detected (Tables 4, 5, 6). In several sites, the ratio of Al
to A2 was nearly 1:1 when totaled over the monitored years. At sites where the ratio was
not close to a 1:1, the A1 CT occurred more frequently when examined across the isolate
totals for a particular site (Tables 4, 5, 6).
Mefenoxam Sensitivity

The SW Michigan region (Allegan Co.) surrounding the river system has a history
of pickling cucumber production (>40 years) and phenylamide fungicide use for disease
management. Each year, river Sites yielded P. capsici isolates of each of the mefenoxam
sensitivity categories (S, IS, and I) (Table 4). Approximately half of the P. capsici
isolates from the river sites in 2002 were intermediately sensitive with one quarter of the
isolates insensitive (Table 4). Most isolates from 2003 and 2004 were insensitive (Table
4). In the last year of monitoring (2005), there was a change in the overall mefenoxam
sensitivity of the pathogen population; most (88%) isolates were sensitive (Table 4).

The NW Michigan region (Oceana Co.) surrounding the two monitored ponds has
a relatively Short history of vegetable production with reports of low reliance on
mefenoxam for disease management. Nearly all P. capsici isolates collected during 2002

to 2005 were sensitive (Table 5). In 2002, there was a single intermediately sensitive

50

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52

Table 6. Summary of P. capsici baiting isolate characterization for Michigan ditches

(Lenawee and Oceana C03), 2004 and 2005.

 

 

 

 

 

 

 

 

 

Mefenoxam (Ridomil Gold)
Sensitivitya
2004 2005

Site CT [S S I IS S Total
1: Run-off A1 9 2 -b - - 14
SE Michigan

A2 3 4 - - - 7
(Lenawee Co.)
2: Culvert A1 - - - 0 7
NW Michigan A2 _ _ _ 0 0 7 7
(Oceana Co.)
Total 6 13 2 0 1 13 35

 

 

 

 

 

 

 

 

aIndicates mefenoxam sensitivity rated as I (insensitive), IS (intermediately sensitive), and
S (sensitive) to 100 ppm mefenoxam.
I’Indicates that specific site was not monitored in that year of study.

53

isolate collected (Table 5). The culvert ditch monitored in 2005 yielded 14 P. capsici
isolates, 13 of which were sensitive to mefenoxam (Table 6). The run-off ditch
monitored in 2004 in SE Michigan (Lenawee Co.) is surrounded by a region that has
history of varied vegetable and agronomic crop production. Mefenoxam has been relied
upon in recent years to manage vegetable diseases. The majority of the P. capsici
isolates collected were intermediately sensitive with approximately 30% also insensitive
(Table 6).
Water Temperature

Water temperatures were Significantly correlated to positive P. capsici detection
from the monitoring Sites from 2002 to 2005 when analyzed using a non-linear quadratic
polynomial regression (R2=0.9408) (Fig. 3). Most (92%) of the baiting periods yielding
positive incidence of P. capsici had water temperatures of 15-25°C (Table 7).
Phytophthora capsici was not identified when water temperatures fell below 14°C or rose
above 25°C (Table 7). Average water temperatures at the river monitoring sites during
baiting periods with positive P. capsici detection were 17 to 19°C. At the pond Sites,
average water temperatures during baiting periods with P. capsici infestation were 20 to
22°C. The run-off and culvert ditch sites had average water temperatures of 21 °C during
baiting periods with positive P. capsici detection.
Rainfall

Rainfall data provided an approximation of the amount of free water present at
water monitoring Sites and nearby fields. However, these values did not reflect the total

precipitation as grower irrigation data was not collected. For the river system, total

54

 

20

18 J R2=0.9408 .
f=y0+a*x+b*x"2

16*
14*

12‘

% incidence
'5
I

 

 

O . I I I I .
5 10 15 20 25 3O

 

Water temperature (°C)

Figure 3. Non-linear, quatratic regression curve of average water temperature (°C)
during baiting periods from all sites and years (2002 to 2005) and the percent incidence

of P. capsici detection.

55

Table 7. Average water temperature (°C) during baiting periods from all Sites and years

(2002 to 2005) and the number of baiting periods with positive detection of P. capsici.

 

 

Temperature Total # of # of baiting % incidence
during baiting baiting periods periods with P.
(0C) capsici
5-10 24 0 0
10-15 114 6 8
15-20 277 47 17
20—25 120 21 18
25-30 8 0 0

 

56

Figure 4. Rainfall (weekly totals) and P. capsici detection data for river
monitoring Sites in Allegan Co., Michigan in A) 2002, B) 2003, C) 2004,
and D) 2005. Gray vertical bars indicate rainfall (mm). Stars indicate

positive P. capsici detection.

57

Rainfall (mm)

160

 

2002 River Sites 1, 2, and 3

120-
* ********

80-

 

0 .3... all

 

 

160
2003 River Sites 1 - 6

1201
1 *iitit a

40-

 

 

 

 

 

 

 

 

._,UU ngDH_UD HHQUH

0 _ ., .
160

 

2004 River Sites 1 and 4 - 6

120‘
*it*** t

80-

40- . '
0lmllflglflmflgflfl_mmmflnmfl__m Uflflfl

160

 

2005 River Sites 1 and 4 - 6

 

 

ZD
120—
* ****
80-
40-
A o o} °o Q 5‘ 4
e o o- c 0
\Q \S \x \Y‘ \CO \0 \‘é
Date

58

 

 

 

rainfall was greatest in 2003 and 2004 (Fig. 4). Rainfall from all years averaged <40 mm
per week during baiting periods with positive P. capsici detection at river sites (Fig. 4).
No Significant rainfall was recorded in 2002 or 2005 at the monitored pond sites in NW
Michigan (Figs. 5). Pond site rainfall was greatest in 2003 and 2004 (Figs. 5). Weekly
rainfall was <30 m during baiting periods with positive detection of P. capsici at the
Lenawee Co. ditch (Fig. 6). No significant rainfall was recorded at the Oceana Co.
culvert ditch during baiting periods in 2005 (Fig. 6).
AFLP Fingerprinting

AF LP analysis provided genetic confirmation of P. capsici identification to
support our morphological determination. Genetic fingerprinting of P. capsici isolates
collected from water monitoring sites from 2002 to 2004 did not indicate an association
between similarity groups and Specific locations, or years collected (Fig. 7). Three
similarity groups (with >80% homology) were resolved fiom a cluster analysis of 56
isolates. Group I contained isolates from river, pond, and ditch sites from 2002 to 2004.
The second group was comprised entirely of isolates from river Sites 1, 3, and 4 from
2002 to 2004. The smallest of the similarity groups, Group III, contained isolates from

the Lenawee Co. ditch site from 2004 (Fig. 7).

59

Figure 5. Rainfall (weekly totals) and P. capsici detection data for pond
monitoring sites in Oceana county, Michigan, A) 2002, B) 2003, C) 2004,
and D) 2005. Gray vertical bars indicate rainfall (mm). Stars indicate

positive P. capsici detection.

()0

Rainfall (mm)

 

160 -
2002 Pond Sites 1 - 3
120 -

80 r

404

*‘ktitit

 

0 r r

 

160
120 J

80-

2003 Pond Sites 1 and 2

 
 

 

120 ‘

 

 

 

 

 

2005 Pond Site 1 D
120 r
i 'k
80 ~
40 -
0 g a m I I H {—1 FT] S 1 I
Q‘ rt?) 05‘ 33 0% 08
NY, \“S O \ x No"
Date

61

 

160

 

 

2004 Run-off Ditch l Lenawee County A
120 a
«k t i i
80 -
:53 160
E 2005 Culvert Ditch 2 Oceana County B
m 120 1
t t a» t t t a
80 -
40 ~
0 am I _m r DD I _ I 1:21 i

 

 

 

\Y~ \VP \8 O \‘c \‘5" \O 5‘

Date

Figure 6. Precipitation (weekly totals) and P. capsici detection data for ditch monitoring
sites in A) Lenawee (2004) and B) Oceana (2005) Cos., Michigan. Gray vertical bars

indicate rainfall (mm). Black stars indicate positive P. capsici detection.

62

Group I

 

 

LTI! C T:MS Year Site
9541AI:S 2002 Pond I

9669 A2:IS 2003 River 1
9681 A2:S 2003 River 1

ll

 

_'—‘19642 A2:S 2003 River I
9715 A2:I 2003 River 1
19572 AI:IS 2002 River I

'9581 AI:S
9539 AI:S
9576 AI:S

 

 

 

 

 

 

Group II

9653 Alzl

2002 River I
2002 River 3
2002 River 3

9577 AI:IS 2002 River I
9570 AI:IS 2002 River I
10439 A1:IS 2004 Ditch l

2003 River 3

10437 A1:IS 2004 Ditch I
I10136 AI:IS 2004 Ditch I
10152 A1:IS 2004 DItch I

 

 

—\0
cu
---w
are.
N

> >
__—

 

111

9565 AI:S
9588 A '
9655 A2:
9672

9737 A2: IS
9688 A2: IS
9649 A1: I
9732 Al: I
9643 Al: I

I10151 Al:lS 2004 Ditch 1
10137 A1:IS 2004 Ditch I
10436 A2:IS 2004 Ditch I

2002 River I
2002 River 1
2003 River 1
2003 River I
2003 River 1
2004 Ditch 1
2002 River 1
2003 River 1
2002 River 1
2003 River I
2003 River I
2003 River 3
2004 River 4
2004 River 4
2004 River 4
2004 River 4
2004 River 4
2003 River I
2003 River I
2003 River 1
2003 River I
2003 River 1
2003 River I
2003 River 1
2003 River I
2003 River I
2003 River I

724 A1:I 2003River1
9730 AI: IS 2003 River]
9670 A1: I 2003 River]
9702 A2:I 2003 River]
9691 A:I 2003 River]
10135 A2:l 2004 Ditch 1
10155 A2: I 2004 Ditch 1
10143 A2: IS 2004 Ditch I
0150 A2: I 04 Ditch l
I,” ,l'HjIfl'llir,‘10147A2:IS20040itch1

0.69 0.77 0.85 0.92 1.00
Similarity Coefficient

 

3 \O\D\O\O©
\IONO\O\GN
(:3me
\OM'JIO
>>>
.":’ N
"5.. —

 

Group III

 

 

 

-11

Figure 7. Cluster analysis of P. capsici isolated from Michigan surface water. Long

term numbers (LT#) refer to culture numbers maintained in the laboratory of Dr. Mary
Hausbeck at Michigan State University. Compatibility Types (CT) are either A1 or A2.
Mefenoxam sensitivity (MS) characteristics are sensitive (S). intermediately sensitive

(IS). or insensitive (I).

63

DISCUSSION

Phytophthora capsici has a broad host range and many disease outbreaks can be
associated with fields with a known history of the pathogen. However, one particular
outbreak and crop loss did not fit into the typical model. New pickling cucumber
growers with established fields on land never planted to P. capsici-susceptible crops
experienced widespread, uniform losses in the SW region of Michigan. Explanations
including movement of the pathogen via equipment seemed inadequate for the sudden,
widespread disease occurring on this new farm. The source of irrigation water (a river)
was suspected as the potential means of pathogen movement. Prior to this research, the
presence of P. capsici in Michigan irrigation water sources had not been reported.

Infested irrigation water is of concern to growers, especially those sharing water
sources such as creeks and rivers. In our study, a Michigan river system in a region with
a long (>40 year) history of cucumber production yielded 211 P. capsici isolates over a 4-
year monitoring period of 6 sites. Initial P. capsici detection in the river typically
followed observation of field disease and a Single or a series of rain and/or irrigation
events. Phytophthora capsici was most frequently detected at monitoring sites with a
susceptible host crop in a nearby field, allowing for run-off of infested water to enter the
river system. The pathogen was detected less frequently from Sites with non—host crops
in nearby fields. However, P. capsici was still present even afier 1-3 years without a host
crop nearby. This pathogen activity may indicate a longer range of movement of P.
capsici within the river system or that there is activity of the pathogen within the
environment outside of host plant tissue. In a study of the movement of a Phytophthora

Sp. (interspecific hybrid of P. cambivora and an unknown Phytophthora related to P.

64

fragariae) on alder in Bavaria, it was determined that once the pathogen was introduced
into a river system upstream, it infected alders downstream (9). In addition, area
nurseries that had high incidence of infected alder seedlings were found to be using water
from infested river courses for irrigation (9). Due to the proximity of the Michigan river
system to susceptible crop production, there are numerous potential sources of P. capsici
infestation. Confirming movement of the pathogen in the river is difficult, as there was
no consistent genetic similarity among isolates from specific sites. With a sexually active
polymorphic population, we cannot state that isolates from upstream have, in fact, moved
downstream using the baiting and AF LP techniques. However, we know that P. capsici
is present in the surface water and that pathogen propagules likely move with water, as
indicated in the Bavarian study (9).

Overhead irrigation with water infested with P. capsici is an effective means of
field inoculation. A research plot at the Michigan State University Muck Soils Research
Farm in Laingsburg, Michigan, was successfully inoculated with P. capsici by injecting
zoospore and sporangial inoculum into the irrigation system. In just two
inoculation/irrigation events, the plot was adequately inoculated, resulting in unifoml and
severe disease symptoms on established pickling cucumber plants (Lamour and
Hausbeck, unpublished results). In the 6 years following initial inoculation of this
research plot, infestation of P. capsici has been persistent. Susceptible crops planted in
this plot have become diseased without additional pathogen inoculation.

The extensive use of phenylamide fungicides for disease management can render
P. capsici field populations insensitive (10, 11, 12). Southwest Michigan has a long

history (>15 years) of P. capsici disease incidence and use of phenylamide fungicides,

65

including mefenoxam. Northwest Michigan has a relatively shorter history of P. capsici
infestation and use of phenylamides. In particular, mefenoxam application in the NW
region has been limited and judicious. Characterization of populations of P. capsici
collected from SW Michigan from 1997-2001 indicated much mefenoxam insensitivity
(12). Isolates from NW Michigan were sensitive to the fungicide (11). This previous
work serves as a historic baseline of population resistance to mefenoxam for our regions
of investigation.

From the river sites in SW Michigan, there was a change in P. capsici isolate
sensitivity to mefenoxam during the 2002 to 2005 growing seasons. In the first
monitoring year (2002), most isolates were intermediately sensitive; in the following 2
years, approximately half the isolates were completely insensitive. The trend toward
insensitivity was not observed in 2005 with all but 10% of isolates sensitive. Previously,
a P. capsici population from SW Michigan did not revert from mefenoxam insensitive to
sensitive during a 2-year crop rotation to non-host crops (12). The isolates collected in
2005 from River (creek) site 1 may have been introduced from a different source. In
other studies with Pythium aphanidermatum, however, it has been noted that metalaxyl-
resistant isolates have declined in number after metalaxyl use was eliminated for at least
3 years. The sensitive isolates of P. aphanidermatum then become more prevalent;
however, not in numbers large enough to allow the use of metalaxyl (22).

Irrigating from ponds also poses a risk of introducing P. capsici infestation.
Pond Site 1 (naturally-fed) yielded P. capsici in each of the four years monitored despite
the planting of non-host crops in the nearby field in 2004 and 2005. One of the two well-

fed ponds yielded just one pathogen isolate over two monitoring years. Ponds fed by

66

deep wells may provide a safer option to growers interested in using surface water for
irrigation with low risk of pathogen infestation, provided that run-off from nearby fields
does not enter the holding pond. A run-off and a culvert ditch yielded P. capsici for
nearly every period monitored. The ditches received direct run-off of water from
numerous infected fields and so had a relatively high potential risk of infestation. Both
ditch sites fed into water retention/pumping ponds used for irrigation.

Water temperature, zoospore concentration, and light may determine the length of
time zoospores can continue to swim and remain viable (2, 3). When lupines were used
to bait for Phytophthora spp. in cranberry irrigation reservoirs in New Jersey, 3
significant positive correlation was observed between water temperature and detection of
P. cinnamomi; a negative relationship was observed for water temperature and P.
megasperma (19). In our study, regression analysis determined that water temperatures
were correlated with P. capsici incidence on pear or cucumber baits (R2=0.9408). In
addition, there appeared to be a lower temperature threshold (14°C) at which P. capsici
was not detected. Phytophthora palmivora and P. citrophthora have been shown to
remain motile in water at an optimal temperature of 17 and 125°C, respectively (2).
Further laboratory studies may be needed to elucidate the relationship between water
temperature and P. capsici zoospore activity. In Michigan, low water temperature
typically occurs at a time when crops may not be established (early spring) or have been
removed (early fall), thereby impacting the presence of P. capsici in surface water.

There was no association between P. capsici similarity groups and specific
locations or years indicating that populations of the pathogen in the monitored river

system were not unique or isolated to specific Sites or years. Phytophthora capsici does

67

not appear to be over-wintering in the water sources, as fingerprints are not monomorphic
across years at individual sites. In addition, positive detection of the pathogen never
occurred prior to mid-July or after late-September, despite water monitoring efforts
which began in April and ended in late October at most Sites.

This study demonstrated the successful use of pears and cucumbers as P. capsici
baits in Michigan surface water. The detection of P. capsici in water during the growing
seasons of 2002 to 2005 appeared to be associated with both incidence of disease in
nearby fields, and history of disease in these fields. The pathogen was detected in water
when non-host crops were planted near monitoring Sites. Monitoring water during the
host crop-growing season indicated that irrigation water was often infested when the need
to irrigate crops was highest, typically in late-July and August. The concurrence of
positive P. capsici detection and need for field irrigation poses a great risk for
dissemination of the pathogen. Characterization of isolates for sensitivity to mefenox am
and CT both assessed the diversity of the P. capsici population present in water and
indicated that isolates insensitive to mefenoxam may be disseminated by irrigation.

Water temperature exhibited some influence on the frequency of isolation of P. capsici
from baits. There was, however, a low temperature threshold for pathogen presence
which may also coincide with cropping and disease cycles. The reliance of surface water
for irrigation of P. capsici-susceptible crops should be minimized, and if possible,

avoided to prevent further pathogen spread.

68

ACKNOWLEDGEMENTS
Funded by Michigan Specialty Crop Block Grant for Pickling Cucumbers: Increase
Marketability of Cucumbers for Pickling by Decreasing Fruit Rot Caused by
Phytophthora and Pythium. Project GREEEN (a cooperative effort by plant-based
commodities and businesses with Michigan State University Extension, the Michigan
Agricultural Experiment Station, and the Michigan Department of Agriculture). Thanks
to Dr. K. H. Lamour and R. Donahoo of University of Tennessee, and J. Woodworth and
K. Cervantes of Michigan State University for technical support. I would also like to
acknowledge the many Michigan grower cooperators who graciously allowed me routine

access to various surface water sources and nearby fields.

69

10.

ll.

LITERATURE CITED

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and Pythium spp. in components of a recycling irrigation system. Plant Disease
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Erwin, D. C., Bartnicki-Garcia, S., Tsao, P. H. 1983. Phytophthora: its biology,
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Erwin, D. C., and Ribeiro, O. K. 1996. Phytophthora diseases worldwide. APS
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Gevens, A. J ., and Hausbeck, M. K. 2004. Phytophthora capsici isolated from
snap bean is pathogenic to cucumber fruit and soybean. Phytopathology 95:8 162.

Gevens, A. J ., Lamour, K. H., and Hausbeck, M. K. 2005. Screening cucumber
germplasm for resistance to Phytophthora capsici using a detached fruit method.
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Habera, L., Smith, N., Donahoo, R., and Lamour, K. 2004. Use of a single
primer to fluorescently label selective amplified fiagment length polymorphism
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Hausbeck, M. K., and Lamour, K. H. 2004. Phytophthora capsici on vegetable
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Hong, C., Richardson, R. P., and Kong, P. 2002. Comparison of membrane
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Jung, T., and Blaschke, M. 2004. Phytophthora root and collar rot of alders in
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Lamour, K. H., and Hausbeck, M. K. 2000. Mefenoxam insensitivity and the
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Lamour, K. H., and Hausbeck, M. K. 2001. Investigating the spatiotemporal

genetic structure of Phytophthora capsici in Michigan. Phytopathology 91 :973-
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Lamour, K. H., and Hausbeck, M. K. 2003. Susceptibility of mefenoxam-treated
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Lamour, K. H., and Hausbeck, M. K. 2003. Effect of crop rotation on the
survival of Phytophthora capsici in Michigan. Plant Disease 87:841-845.

Larkin, R. P., Gumpertz, M. L., and Ristaino, J. B. 1995. Geostatistical analysis
of Phytophthora epidemic development in commercial bell pepper fields.
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Mitchell, D. J ., and Kannwischer-Mitchell, M. E. 1992. Phytophthora. Pages 31-
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L., Mihail, J. D., Rush, C. M., eds. APS Press. St. Paul, Minnesota.

Neher, D., and Duniway, J. M. 1992. Dispersal ofPhytophthora parasitica in
tomato field by furrow irrigation. Plant Disease 76:582-586.

Noon, J. P., and Hickman, C. J. 1974. Oospore production by a Single isolate of
Phytophthora capsici in the presence of chloroneb. Canadian Journal of Botany
52:1591-1595.

Oudemans, P.V. 1999. Phytophthora species associated with cranberry root rot
and surface irrigation water in New Jersey. Plant Disease 83:251-258.

Ristaino, J. B., and Johnston, S. A. 1999. Ecologically based approaches to
management of Phytophthora blight on bell pepper. Plant Disease 83: 1080-1 089.

Uchida, J. Y., and Aragaki, M. 1980. Chemical stimulation of oospore fomiation
in Phytophthora capsici. Mycologia 72:1103-1108.

Vargas, J. M., Jr. 2004. Management of turfgrass diseases, Third Edition. Wiley
Publishing, John Wiley & Sons, Inc., Hoboken, New Jersey.

Vos, R, Hogers, R., Blecker, M., Reijans, M., van der Lee, T., Homes, M.,
Frijters, A., and Pot, J. 1995. AF LP: A new technique for DNA fingerprinting.
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Waterhouse, G. 1963. Key to the species of Phytophthora de Bary.

Mycological Papers, No. 92, Commonwealth Mycological Society, Kew, Surrey,
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25. Yamak, F., Peever, T. L., Grove, G. G., and Boal, R. J. 2002. Occurrence and
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the Wenatchee River valley of Washington State. Phytopathology 92:1210-1217.

72

CHAPTER II
IDENTIFICATION AND CHARACTERIZATION OF PH Y T OPHTHORA

CAPSICI ON BEAN (PHASEOLUS VULGARIS) IN MICHIGAN

73

ABSTRACT

Commercial bean fields were observed with water-soaked foliage, stem necrosis,
and overall plant decline along the surface water drainage pattern in 3 Michigan counties.
All four fields have history of Phytophthora capsici infestation. Diseased tissue from
stems, petioles, leaves, and pods collected from the fields yielded a total of 680 isolates
of P. capsici. No isolates were recovered from bean roots. Koch’s Postulates were
completed with representative pathogen isolates collected in 2003, confirming P. capsici
as the pathogenic organism. All representative isolates were also pathogenic on
cucumber fruit. The majority of P. capsici isolates collected were sensitive to the
fungicide mefenoxam and were of the Al compatibility type. Under laboratory
conditions, six select P. capsici isolates from snap bean (2003) were all pathogenic on
twelve bean types, including soybean, causing water-soaked lesions, necrosis, and
wilting. Disease on beans was rated (0=healthy, 5=dead) at 6 days post inoculation and
ratings averaged 24.0. A group of 131 isolates from 2003 and 2004 were subjected to
amplified fragment length polymorphism (AF LP) analysis to investigate genetic diversity
among isolates and geographical populations. This is the first documentation of P.
capsici on snap and wax beans in MI. Rotating beans with other susceptible hosts is not

currently recommended.

74

INTRODUCTION

Phytophthora blight caused by the fungal-like Oomycete Phytophthora capsici
[Leonian] causes foliar blighting, root, crown, and fruit rot on solanaceous and cucurbit
crops worldwide (1, 11, 16). Disease symptoms may be first observed in the Spring when
seedlings exhibited damping-off following infection by overwintering oospores (18, 19).
Symptoms can occur throughout the growing season as environmental conditions
promote the development of wind and splash-dispersed Sporangia and zoospores (5).
Crown rot results in irreversible wilt and plant death. Lesions on the foliage and the fruit
appear water-soaked and may quickly enlarge. Over time, infected fruit may exhibit a
powdered-sugar appearance from accumulation of sporangia and/or oospores (17).

In Michigan, P. capsici is a limiting factor in cucumber, squash, zucchini, pepper,
and pumpkin production despite adherence to recommended control strategies, including
crop rotation, surface water management, and fungicide application (9). The fungicide
mefenoxam was typically used to manage P. capsici, however, development of
insensitivity of pathogen populations has limited its usefulness (17, 20, 21, 25).
Reversion to mefenoxam sensitivity in field populations was not observed within a 2-year
monitoring period when the fungicide selection pressure was removed (19, 20).

While beans are known to be susceptible to Phytophthora spp. such as P. sojae, P.
megasperma, P. nicotiana, and P. phaseoli, they have not been included in the host range
of P. capsici (5). Beans have historically been recommended as a suitable rotation crop
with P. capsici-susceptible vegetables. Laboratory inoculations of P. capsici on beans,

lima beans, and soybeans resulted in resistant plant responses in a 1967 study (22).

75

However, in 2000 and 2001, P. capsici was detected on lima beans in commercial fields
in Delaware, Maryland, and New Jersey (2). In Michigan, snap beans were first
diagnosed with P. capsici in 2003 from commercial fields with a history of zucchini
cropping and P. capsici infestation (6, 9).

The pathogenicity and phenotypic characteristics of P. capsici isolates within
field populations are diverse (12, 23, 24). The concept of host specificity of P. capsici
has been explored; however, studies have been restricted to fewer than 30 isolates of
limited geographical diversity (24, 27). In addition, these studies have relied on a single
inoculation technique of delivering zoospores to the soil of seedling differentials (24, 27).
Susceptibility of plants to P. capsici is greatly impacted by method of inoculation,
inoculum density, and environmental conditions (14, 23).

The biology and epidemiology of P. capsici on bean (Phaseolus spp. and Glycine
max) is not known. In Michigan and the north-Atlantic vegetable production regions, P.
capsici is an important disease on many crops (2, 9). Once fields become infested with
P. capsici, disease may occur in subsequent years when a suitable host is planted and
when favorable environmental conditions occur. With the identification of a new host
family (Leguminosae), which includes many common rotational crops, it is important to
elucidate the etiology of this disease on bean for development of appropriate control
strategies. Phenotypic characterization of P. capsici isolates from bean can indicate
diversity of population and firngicide sensitivity, which may aid in management.
Evaluation of the genetic structure of the pathogen population may also detect
subdivisions among populations within a particular area and can reveal host or tissue

specificity. Our research objectives included 1) confirming pathogenicity of P. capsici

76

isolated from beans, 2) characterizing the isolates through phenotypic and genotypic
analyses 3) assessing the range of bean type susceptibility, and 4) comparing virulence of

select isolates on cucumber fruit and soybean.

MATERIALS AND METHODS
Sample Collection and Pathogen Identification

Symptomatic and asymptomatic bean plants were collected from commercial
bean fields in Michigan, including 2 fields (#1 and #2) in Oceana County (Co.) in 2003, 1
field from Cass Co. in 2004, and 1 field from Van Buren Co. in 2005. Fifty to 200 plants
with a range of disease symptoms (from healthy to dead) were collected from each of the
4 fields. Most of the plant samples were taken from areas of the fields with evident
disease symptoms of water-soaked foliage and wilting. Asymptomatic plants
(approximately 25% of all plants collected) were also obtained for analysis, and were
collected by moving through the field in an ‘X’ configuration and randomly harvesting
bean plants.

Leaf, petiole, stem, crown, and root tissues from symptomatic and asymptomatic
bean plants were excised, surface sterilized, and plated onto BARP (benomyl, ampicillin,
rifampicin, and pentachloronitrobenzene)-amended, unclarified V8 juice agar plates (20).
After 3 days of incubation at 24°C under fluorescent lighting at room temperature,
microbial growth was assessed by microscopy. Cultures that were morphologically
identified as P. capsici based on characteristics described in the Phytophthora spp. key
by Waterhouse, 1963 (31) were transferred to new BARP plates and single-zoospore

cultures were generated (1 7).

77

Koch’s Postulates and Pathogenicity Screen

Single-zoospore (axenic) cultures of P. capsici were used to inoculate 10, 3-week-
old snap bean plants (‘HyStyle’) with 7-mm diameter mycelial/sporangial agar plugs on
leaf and petiole tissue. After symptoms developed, 5-7 days post inoculation (dpi), tissue
at the margin of healthy and diseased was excised, surface sterilized, and plated onto
BARP for incubation at 24°C under fluorescent lighting at room temperature for 3 days.
Based on morphological characteristics (as stated above), the organism was identified as
P. capsici and confirmation of pathogenicity on bean was completed. Bean plant
inoculations to fulfill Koch’s Postulates were conducted two times.

Further pathogenicity testing was conducted on cucumber fruit that were
produced at the Plant Pathology Farm at Michigan State University, East Lansing, MI, in
a field with no history of P. capsici infestation. Fruit were surface disinfested with
sodium hypochlorite (5%), rinsed with sterile water, and dried under ambient laboratory
conditions (21:1:2°C). A 7-mm mycelial/sporangial agar plug from each of the 20 P.
capsici isolates from snap bean (Oceana Co. #1) was placed on the surface of intact fruit,
covered with a microcentrifuge tube, and sealed to the fruit with petroleum jelly (7).
Fruits were incubated in trays covered with plastic wrap to maintain high humidity.
Disease was assessed by measuring 3 lesion parameters: 1) the diameter of the water-
soaked region at the leading edge of the lesion, from this point forward referred to as
lesion diameter; 2) diameter of the sporulating region (white and powdery) of the lesion,
referred to as Sporulation diameter; and 3) density of pathogen sporulation (relative
number of sporangia/cm2 fruit surface) rated as 0=none, 1=light, 2=moderate, 3=high,

referred to from this point forward as sporulation density. Fruit tests were conducted two

78

times and results averaged. All fruit disease ratings were taken at 3 days post
inoculation. Means were separated and data was analyzed using Tukey’s Studentized
LSD Test at a=0.05 in SAS (SAS Institute, Cary, NC, USA).
Phenotypic Characterization

Compatibility Types: Seven-mm diameter mycelial/sporangial agar plugs from
the edge of expanding single-zoospore-derived cultures were placed at the center of V-8
plates 2 cm away from plugs of isolate OP97 (Al) and SP98 (A2), and incubated at 24°C
in the dark for 7 to 10 days. OP97 and SP98 are P. capsici isolates collected from
Michigan cucurbits which are maintained in the laboratory of Dr. Mary Hausbeck of the
Department of Plant Pathology at Michigan State University. OP97 and SP98 have been
confirmed to be A1 and A2 using American Type Culture Collection standards. CTS
were determined by assessing the presence or absence of oospores between the 2 agar
plugs by microscopy (19).

Mefenoxam Sensitivity: Seven-mm diameter mycelial/sporangial agar plugs from
the edge of expanding single-zoospore-derived cultures were plated onto V-8 agar and V-
8 agar amended with 100 ppm mefenoxam. Plates were incubated at 24°C for 3 days, and
colony diameters measured. Percent grth of an isolate on amended agar was
calculated by subtracting the inoculation plug diameter (7 mm) from the diameter of each
colony and dividing the average diameter of the amended plates by the average diameter
of the unamended control plates. Isolates were assigned mefenoxam sensitivities based
on percent grth of the control. An isolate sensitive (S) to mefenoxam was <3 0% of

control, an intermediately sensitive (IS) isolate was 30-90% of control, and an insensitive

79

(I) isolate was >90% of the control (21). All tests were conducted twice and results
averaged.
Susceptibility of Bean Types to P. capsici

Twelve bean types (Table 1) were either purchased from a commercial seed
supplier or were donated by grower cooperators. Three-week-old bean plants were
inoculated with six P. capsici isolates. Isolates 9948, 9951, and 9974 obtained from
Oceana Co. #1 and isolates 9986, 9988, and 9989 from Oceana Co. #2 were chosen. The
6 isolates selected were representative of unique phenotypic groups based on their
symptom morphology on cucumber fruit and on V-8 agar. Mycelial agar plugs (7—mm
diameter) of l-week-old, single-zoospore-derived cultures were placed onto bean leaf and
petiole tissue, and covered with microcentrifuge tubes sealed with petroleum jelly. One
plug was applied to the middle portion of the surface of a leaf and a second to the portion
of the petiole which joins the main stem on each plant. Individual plants were enclosed
in a sealed plastic bag with a wet paper towel to maintain high humidity. Plants were
incubated under ambient laboratory conditions at 21i2°C and rated for disease 6 dpi. The
disease rating for leaves was: 0=no disease, 1=<25% necrotic, 2=<50% necrotic, 3=50%
necrotic, 4=>50% necrotic, and 5=100% necrotic. The disease rating for petioles was:
0=no disease, 1=<50% petiole necrotic, 2=100% petiole necrotic, 3=100% petiole
necrotic and 25% leaf necrotic, 4=100% petiole necrotic and 50% leaf necrotic, and
5=100% petiole and leaf necrotic. Each test was conducted three times. Disease ratings
were analyzed by mean separation and significant differences were distinguished using

Tukey’s Studentized LSD (or=0.05) in SAS (SAS Institute, Cary, NC, USA).

80

Virulence Comparison of Select Isolates
Six select P. capsici isolates (listed above) were compared for virulence on 3-week-old
soybean plants. A randomized complete block design with four repetitions of each of the
six P. capsici isolates plus control was used. Inoculations and disease ratings were made
as described above for bean plants. The isolates were also compared for virulence on
cucumber fruit. Methods of inoculation and disease rating for cucumber fruit are
described above in pathogenicity screening. Soybean plant and cucumber fruit disease
ratings were analyzed by mean separation and significant differences were distinguished
using Tukey’s Studentized LSD (a=0.05) in SAS (SAS Institute. Cary. NC, USA).
DNA Extraction and AFLP Analysis

Genomic DNA was extracted from approximately 10 mg of freeze-dried P.
capsici mycelium using DNeasy® mini-kits (Qiagen, Valencia, CA., USA). Mycelium
was grown in antibiotic-amended V8 broth and DNA was quantified on an agarose gel
with known standards. EcoRI was obtained from Invitrogen (Carlsbad, CA., USA),
while Msel and T4 DNA ligase were obtained from Takara (Madison, WI, USA). EcoRI
and Msel adapters and primers for ligation and amplification reactions were obtained
from Integrated DNA Technologies (Coralville, IA, USA); Sequence information for the
adapters and primers is published in Vos et al. (30). The fluorescently labeled primers
were obtained from Proligo (Boulder, CO, USA) and were comprised of the EcoRI core
sequence with and without selective nucleotides and either the WellRED D4-PA label
(Proligo) or the WellRED D3-PA label at the 5’ end.

Restriction, ligation, preamplification, and selective amplification reactions were

carried out as described by Habera et al. (8). Fluorescent products from the selective

81

Table 1. Bean types included in laboratory P. capsici pathogenicity screen.

 

 

 

 

 

 

 

Genus species Cultivar
Phaseolus lunatus ‘Bush Lima’
Phaseolus vulgaris var. humilis ‘Fordhook Standard’
‘Bush Tenderpod’
‘Bush Contender’
Phaseolus vulgaris var. vulgaris ‘Pole Bean’
‘Kentucky Wonder Pole’
Phaseolus vulgaris ‘Cranberry Soup Bean’
‘Gold Mine Wax’
‘Black Turtle’
‘Blue Lake White Seed’
Glycine max Soybean

 

 

82

 

amplifications were analyzed on a CEQTM 8000 Genetic Analysis System (Beckman

Coulter, Fullerton, CA, USA) using the manufacturer’s protocols.

RESULTS

All bean fields sampled had history of P. capsici-susceptible crop production and
history of disease on those crops due to P. capsici. Overall symptoms and field disease
pattern on snap and wax beans included localized pockets of plant wilting and death
typically along the surface water drainage pattern (Fig. lA-D). Symptomology included
water-soaking, necrosis, and wilting of stem, petiole, and leaf tissue (Fig. 2). In 2004 and
2005, symptomology also included water-soaking, necrosis, shriveling, and pathogen
sporulation on pods (Fig.3). Disease symptoms were not observed on roots and P.
capsici was not isolated from excised root tissue.

The Oceana Co. fields yielded 49 P. capsici isolates (Table 2). Most (>85%) of
the P. capsici isolates collected from snap bean in this county were from stem tissue
(Table 3). Leaf symptoms included water-soaking and wilting (Fig. 2A,B). Pod tissue,
in 2003, did not exhibit disease symptoms; asymptomatic pods did not yield P. capsici.
In August 2004, disease symptoms similar to those from 2003 were observed in one
commercial snap bean field in Cass Co., Michigan (Fig. 1A,B). Extensive field sampling
resulted in the collection of 227 P. capsici isolates (Table 2). Stem, leaf, and pod tissues
(Fig 3A,B) all yielded P. capsici, with most of the isolates from stem tissue (Table 3).
This was the initial finding of P. capsici on bean pod tissue in this Michigan study. In
August 2005, approximately 300 acres of yellow wax beans were identified with disease

caused by P. capsici in Van Buren Co. in August 2005. Field symptoms of localized

83

 

Figure 1. Field symptoms of P. capsici on bean. A, B) Snap bean field from Cass
Co., Michigan in August 2004. C, D) Yellow wax bean field from Van Buren Co.,
Michigan in July 2005. A-D) Note localized circular pattern of plant decline and
death, and association of plant decline with the surface water drainage pattern.

(Images in this dissertation are presented in color).

84

Figure 2. Symptoms of P. capsici on bean leaves, petioles, and stems. A)
Snap bean, ‘HyStyle,’ from Oceana Co., Michigan, 2003. Note water-
soaking on leaves. B) Snap bean, ‘HyStyle,’ leaf from Cass Co., Michigan,
2004. Note water-soaked leaf symptoms observed on leaf underside. C-G)
Yellow wax bean, ‘Sunrae,’ from Van Buren Co., Michigan, 2005. C)
Circular necrotic lesion observed from leaf underside. D) Dark brown,
necrotic leaf and petiole lesions. E) Necrotic symptoms spreading from
junction of leaf and petiole. F) Brown, necrotic stem symptoms. G) Close
up of stem lesion with pathogen sporulation on necrotic tissue. (Images in
this dissertation are

presented in color).

85

 

86

 

Figure 3. Symptoms of P. capsici on bean pods. A,B) Infected pods of snap bean,
‘HyStyle’, from Cass Co., Michigan, 2004. A) Lesions on pods are dark brown and
sunken. B) Note brown, water-soaking symptoms on pod tip where pod made
contact with the soil. C,D) Infected pods of yellow wax beans, ‘Sunrae,’ from Van
Buren Co., Michigan, 2005. D) Close up of infected pod tip. Note water-soaking
and white pathogen sporulation on surface of necrotic pod tissue. (Images in this

dissertation are presented in color).

87

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530E :25 com 3955335 55 b16858 8325.58 28 C18 59C.

88

Table 3. Analysis of P. capsici isolates identified from commercial bean fields in
3 Michigan counties during 2003 to 2005 by specific plant tissues. No P. capsici was

identified on root tissue.

 

 

 

 

 

 

Year/County Total # of Total # of # of P. capsici isolates from specific
plants P. capsici plant tissues
sampled isolates actual # (% of total)
stem leaf pod

2003/Oceana 50 20 17 (85%) 3 (15%) 0
Field 1

2003/Oceana 50 29 29 (100%) O 0
Field 2

2004/Cass 100 227 93 (41%) 59 (26%) 75 (33%)
2005N an 200 404 137 (34%) 43 (11%) 224 (55%)
Buren

 

 

 

 

 

 

 

 

89

plant decline were similar to those observed in 2003 and 2004 (Fig. 1C,D). Sampled wax
beans yielded 404 P. capsici isolates (Table 2) from stem, leaf, and pod tissues (Figs. 2C—
G and 3C,D). Symptoms on wax bean pods were much more severe and frequent than
those observed on snap bean in 2004. More than half (55%) of the isolates collected
were from infected pods (Table 3).

The average air temperature in Oceana and Cass Cos. during the late-July 2003
and early-August 2004 bean epidemics was 18°C. In late-July 2005, the average air
temperature in Van Buren Co. during the period of wax bean infection was 23°C. Under
laboratory conditions, P. capsici disease on bean could not be initiated at temperatures
>23°C. For this reason, all inoculations on bean plants were carried out under ambient
laboratory conditions (21:1:2°C).

Phytophthora capsici was positively identified based on morphological
characteristics (26) and AF LP analysis. Other organisms that were isolated from both
symptomatic and asymptomatic bean plants included Pythium spp., Alternaria spp.,

F usarium spp., and Phytophthora nicotiana. F usarium spp. were particularly common
from root tissue isolations. On symptomatic plants with positive detection of P. capsici,
other organisms were rare.

Koch’s Postulates and Pathogenicity Screen

Snap bean isolates from Oceana Co. #1 and #2 were pathogenic on ‘HyStyle’ snap beans
and on ‘Fanfare’ cucumber fruit. Disease symptoms on laboratory inoculated bean plants
were identical to those observed in commercial fields: water-soaked foliage, petiole and
stem necrosis, and wilting. Cucumber fruit symptoms from bean isolates included water-

soaking, and pathogen sporulation (Fig. 4) and were identical to symptoms from fruit

90

0988

l)€)5;l

9989

‘1‘)?itv

 

Figure 4. Variation in cucumber fi'uit lesions (at 4 days post inoculation) from
inoculation with 6 select P. capsici isolates from Oceana Co. Michigan snap bean,
2003. A—C) Lesions from P. capsici from Field 1. Lesions vary from A) sunken

and covered in dense pathogen sporulation, which remains appressed to fi'uit

surface, to B) fluffy with dense aerial mycelium. C) Lesion of large diameter with
fluffy aerial mycelium. D-E) Lesions from P. capsici isolates from Field 2. Lesions
with D) moderate and E) dense aerial mycelium. F) Lesion of largest diameter
(when compared to A-E) and moderate appressed pathogen sporulation. Numbers

in the lower right-hand comers of images indicate the isolate number. (Images in this

dissertation are presented in color).

91

inoculation with cucumber isolates. Phenotypic variation in sporulation density and
aerial mycelium morphology was observed on inoculated cucumber fruit (Fig. 4). Isolate
9974 produced fruit lesions that were water-soaked and sunken with dense, appressed
pathogen sporulation (Fig. 4A). Isolates (9951 and 9989) (Fig. 4B,E) produced fruit
lesions with aerial mycelial and sporangial production that appeared white and fluffy,
while lesions from isolates 9948, 9988, and 9986 (Fig. 4C,D,F) were of larger diameter

and exhibited ample pathogen sporulation with limited aerial mycelium.

Phenotypic Characterization

Isolates of P. capsici from Oceana Co. #1 and #2, and Van Buren Co. were
primarily of the A1 CT (Table 2). Cass Co. isolates, however, were more than 75% A2
CT (Table 2). Greater than 80% of P. capsici isolates from Oceana Co. #1 and #2 were
sensitive to the fungicide mefenoxam (Table 2). Fewer isolates from Cass and Van
Buren Cos. were sensitive to mefenoxam; intermediately sensitive isolates comprised
>40% of the population sampled, with 20-32% insensitive to mefenoxam (Table 2). Cass
and Van Buren Cos. have a history of pickling cucumber production, P. capsici incidence,
and frequent mefenoxam use.
Susceptibility of Bean Types to P. capsici

A11 12 bean types (Table 1) inoculated with six select P. capsici isolates (listed
above) resulted in water-soaking and necrosis of both leaf (Fig. 5) and petiole tissue.
Bean types exhibited equal susceptibility to P. capsici; no significant differences in
disease were determined among bean types (data not shown). On petioles, disease spread

rapidly into leaves and stems, resulting in necrosis and plant wilting in just 6 dpi. Under

92

 

Figure 5. Localized water-soaked foliar lesions on bean types caused by

P. capsici from Oceana Co., Michigan snap bean in 2003 (Field 1).

Lesions are depicted at 5 days post inoculation under laboratory conditions.
Symptoms of localized water-soaking and necrosis are evident on A) ‘Bush
Tenderpod,’ B) soybean (leaf underside), C) ‘Bush Lima,’ and D) soybean

(leaf surface). (Images in this dissertation are presented in color).

93

experimental conditions, the pathogen did not sporulate on bean tissues. It was important
to maintain the inoculated plant under conditions of high humidity to initiate disease.

Virulence Comparison of Select Isolates

On cucumber fruit, isolates exhibited significant differences for overall lesion
diameter, sporulation diameter, and sporulation density (Fig. 6), however, all isolates
were highly virulent. For each of these parameters, isolate 9988, from Oceana #2, was
consistently the most virulent (Fig. 6). Isolates 9948 and 9951, both from Oceana #1,
were consistently rated with the lowest average disease (Fig. 6).

All isolates were also virulent on soybean. Soybean petioles inoculated with the
six select P. capsici isolates exhibited significantly different disease ratings. However,
isolate 9948 (Oceana #1) was most virulent on soybean petioles, with isolates 9988 and
9989 (both from Oceana #2) exhibiting the least virulence (Fig. 7). Inoculated soybean
leaves did not yield significant differences among the six P. capsici isolates (Fig. 7).
DNA Extraction and AFLP Analysis

Fingerprint analysis of 131 P. capsici isolates (from 2003 and 2004) from snap
bean indicated that there was genetic similarity of isolates from individual counties and
specific years. Groups I and 11 contain isolates from Cass County (Fig. 8). Within Group
I there are 15 subgroups which exhibit 100% similarity based on AF LP analysis; in
Group II, just 5 100% similarity subgroups (Fig. 8). Group III contains isolates from
Oceana Co. #1 and #2 (Fig. 8) and is comprised of just 3 100% similarity subgroups (Fig.
8). Of the 24 Cass Co. isolates fingerprinted, 14 (58%) were unique (Fig. 8). There were

37 (35%) unique isolates from Van Buren Co. (Fig. 8). There does not appear to be a

94

Figure 6. Comparison of disease ratings (sporulation density, sporulation
diameter, and lesion diameter) for 6 select P. capsici isolates from

snap bean on cucumber fruit. Isolates 9951, 9948, and 9974 are from
Field 1 Oceana Co., 2003. Isolates 9989, 9986, and 9988 are from Field 2
Oceana Co., 2003. Ratings were taken at 3 days post inoculation.

Letters indicate significant differences in disease among isolates using

Tukey’s Studentized Range Test at (1:0.05.

95

 

 

 

 

 

 

u u _ -

4 3 2 .I. 0 oo 6 4 2 0
9-8 55qu nougdomm 35v 55:55 comes—Beam

 

 

006420

35V 55:85 5554

9951 9948 9974 9989 9986 9988

control

P. capsici Isolate

96

_ Petiole

5'0 C Leaf

=death)

Disease Rating

(0=no disease 5

 

9951 9948 9974 9989 9986 9988

P. capsici Isolate

Figure 7. Average disease ratings of 6 P. capsici isolates from snap bean on soybean
leaves and petioles. Isolates 9951, 9948, and 9974 were from Field 1, Oceana Co.,
Michigan, 2003. Isolates 9989, 9986, and 9988 are from Field 2, Oceana Co., Michigan,
2003. Significant differences in petiole disease among isolates are indicated by lower
case letters within black vertical bars. Significance was determined using Tukey’s
Studentized Range Test at a=0.05. No significant differences were found in leaf disease

among P. capsici isolates. Control plants exhibited no disease.

97

 

 

 

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Figure 8. Cluster analysis of P. capsici isolated from Michigan snap bean. Long Term
Numbers (LT#) refer to culture identification numbers. Compatibility Types (CTs) are
either A1 or A2. Mefenoxam Sensitivity (MS) characteristics are sensitive (S),

intermediately sensitive (1S), or insensitive (I).

LT# C T:MS Year Site

 

 

 

 

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ceana
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ceana 2

Table 4. Bean plant tissue diversity of AF LP similarity groups of P. capsici
isolates from Oceana and Cass Cos. Michigan in 2003 and 2004. Cluster groups
exhibit approximately 80% homology. Similarity subgroups exhibit 100%

homology based on AF LP analysis.

 

Total # of

Bean plant tissue isolates in

Cluster Similarity similarity
group subgroup pod stem leaf subgroup

 

Group I

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10

Group II

Group 111

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275 300 325 350 375 400 425 450
Size (nt)

Figure 9. A-D) Segments of electropherograms from amplified fragment
length polymorphism (AFLP) profiles of genomic DNA from four P. capsici
isolates recovered from pumpkin and snap bean in Michigan. The AF LP
profiles were produced using a Beckman-Coulter CEQ capillary genetic
analysis system with the primer pair E-AC/M-CA and visualized using the CEQ
fragment analysis software. Arrows indicate polymorphic markers of 325, 362.

415, and 452 nucelotides.

lOl

genetic subdivision in the population of P. capsici based on plant tissue or host. Each of
the 23 100% similarity subgroups contains P. capsici isolates derived from multiple bean
plant tissues (Table 4). Segments of electropherograms from AF LP profiles of genomic
DNA indicate that a P. capsici isolate from pumpkin (Oceana Co., Michigan, 1997) has
homology to isolates from snap bean from both Cass and Van Buren Cos., Michigan (Fig.
9). The selection of the 3 bean isolate profiles is representative of the total 131

fin gerprinted.

DISCUSSION

The discovery of foliar and pod blighting of bean caused by P. capsici in
Michigan is alarming. This pathogen poses a potential threat to a range of bean types
within the family Leguminosae. Michigan produces approximately 2.2 million acres of
beans (snap beans, dry beans, and soybeans) for both processing and fresh market, which
may be susceptible (15). Our research goals were to phenotypcially and genotypically
characterize P. capsici isolates from bean, and to assess P. capsici pathogenicity and
virulence on additional bean types and cucumber fruit. From these objectives we can
make inferences about the etiology of P. capsici on bean in Michigan.

The biology and epidemiology of P. capsici on cucurbit and solanaceous crops
has become well documented in recent years (9, 17-21). In a spatiotemporal study of P.
capsici in a Michigan commercial field, it was determined that a P. capsici epidemic on
squash in 1999 was initiated by dormant oospores generated 5 years previously, despite
rotation to corn and soybeans (18). The survival of pathogen oospores in soils for long

periods of time indicates that seasonal introduction of the pathogen to the field is not

102

 

 

 

necessary for subsequent disease initiation. Therefore, the introduction of P. capsici in
Michigan bean fields in 2003 to 2005 was likely due to resident soil populations.

Genetic fingerprinting of P. capsici isolates from snap beans in Michigan
indicated the absence of host specificity or pathogen population subdivision based on
host. Fingerprints of representative isolates from bean indicate homology with an isolate
from pumpkin. In addition, all bean isolates were pathogenic on cucumber fruit causing
symptoms identical to those isolated from cucurbit hosts. The snap bean isolates are not
unique to bean, but are part of resident populations in these fields that have the potential
to infect susceptible crops. It has been documented that P. capsici field populations
include isolates with a range of diversity in phenotypic characteristics; however,
genotypic diversity of the representative isolates from the Michigan counties in 2003 and
2004 was minimal. The pathogen populations were also not divided based on plant
tissue; similarity subgroups contained isolates from bean pod, stem, and leaf tissue.

Phytophthora capsici spread is limited to short geographical distances, typically
within a field (9), however, infested irrigation surface water may also provide a source of
longer distance pathogen dissemination (9). History of susceptible cucurbit cropping in
the studied bean fields indicates that P. capsici had become an annual concern with
increasing severity (18, 19). Although P. capsici on bean presents new management
challenges regarding crop rotation and control options, we are faced with an old pathogen
on a new host enabling us to draw from recent studies on established host crops to further
understand pathogen epidemiology.

Bean disease symptoms caused by P. capsici were only detected on above-

ground plant tissues and root sampling yielded no P. capsici. In addition, root disease

103

was not detected when plants were inoculated with a zoospore soil drench under
laboratory conditions; disease could only be attained by direct foliar inoculation. In some
cases, plant root exudates are essential to chemotactically attract zoospores for
encystment to occur (4, 5). Perhaps these exudates are limited or absent in bean roots and
are therefore not as readily susceptible. Other environmental or plant resistance factors
may also be involved in this phenomenon. During disease epidemics of cucurbit and
solanaceous crops, the ideal range of air temperature for P. capsici is 25-30°C (9). Lower
average air temperature was required for disease initiation and progress on bean hosts.
Water is also an important factor in disease initiation and development for cucurbit and
solanaceous hosts of P. capsici. In our study, bean plants inoculated with P. capsici
required high humidity, however, little is known about the effect of water on the
interaction of P. capsici on leguminous hosts under field conditions.

Compared to P. capsici disease on green snap beans in 2003 and 2004, disease on
yellow wax beans in 2005 was markedly worse, with higher pod incidence and severity.
The large-acreage yellow wax bean field was situated adjacent to green ‘Romano’ beans
which exhibited no disease symptoms and did not yield P. capsici when sampled.

Further examination of cultivar susceptibility is necessary to determine what factors may
be responsible for P. capsici resistance in bean.

The presence of both CTS in all bean fields studied indicated that sexual
recombination is likely occurring and with it, genetic variation within the population.

In Oceana Co., isolates were predominantly sensitive to mefenoxam suggesting that
although these 2 fields have had application of the fungicide in their management

programs, use has been judicious. Lamour and Hausbeck studied a population of P.

104

capsici from Oceana Co. from 1998 to 2000 (19); all but one of the isolates examined
were sensitive to mefenoxam. Isolates from Cass and Van Buren Cos. exhibited some
sensitivity to mefenoxam, however a large percentage of the populations were
intermediately and fiilly insensitive to the fungicide. The Cass and Van Buren Co. bean
fields are known to have had extensive pickling cucumber production in their recent
history. The fields were rotated to bean production as a means to avoid and/or limit crop
losses to P. capsici, as pathogen populations were high and mefenoxam was having
reduced efficacy.

Under laboratory conditions, all 6 snap bean P. capsici isolates were pathogenic
on the 12 bean types inoculated, including soybean, indicating that most bean types
grown in rotation with cucurbit or solanaceous crops may be at risk. Although some
significant variation in virulence was observed among snap bean isolates on soybean
petioles, all isolates were highly aggressive on both leaf and petiole tissues. This petiole
response is likely the result of plant structural properties (all petioles are not of equal
length and diameter). All isolates were also pathogenic on cucumber fruit and exhibited
significant differences in disease severity.

Due to an increase in agricultural land infested with P. capsici, it is not
economically viable for growers to simply remove fields fi'om host crop production.
Instead, they must recognize the biology of the pathogen and focus on factors such as
cultural control and water management (9). Commercial bean production is, on average,
less profitable than solanaceous or cucurbit crop production, for this reason, fungicide
applications may not be an economically viable option for bean disease management.

Future evaluation of bean cultivar resistance may provide additional options for

105

continued bean production in infested fields. Avoiding the introduction of P. capsici into
uninfested or new fields is the key management strategy. In the past, rotation with a bean
cultivar was an option for growers with infested fields, however, this practice is no longer

recommended (9).

ACKNOWLEDGEMENTS
I gratefully acknowledge our grower cooperators, Dr. K.H. Lamour and R. Donahoo for
assistance with AF LP work and analysis, and KL Cervantes for technical assistance with
cultures. In addition I acknowledge the support of the MDA Michigan Specialty Crop
Block Grant for Pickling Cucumber; Pickle Seed Research Fund, Pickle Packers
International, Inc.; Pickle and Pepper Research Committee of MSU, Pickle Packers
International, Inc.; Project GREEEN (a cooperative effort by plant-based commodities
and businesses with Michigan State University Extension, the Michigan Agricultural
Experiment Station, and the Michigan Department of Agriculture): Developing strategy
to limit Phytophthora diseases on vegetables; and Gerber Products Company: Managing
diseases of snap beans 2003-2004. In addition, I would like to acknowledge Mr. James

K. Jordan for donation of soybean seed.

106

10.

11.

12.

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Nucleic Acids Res. 23:4407-4414.

Waterhouse, G. M. 1963. Key to the species of Phytophthora de Bary.

Mycological Papers, No. 92, Commonwealth Mycological Society, Kew, Surrey,
England. 22 pp.

109

CHAPTER III
DEVELOPMENT OF A DETACHED CUCUMBER (C UC UMIS SA TIVUS) FRUIT
ASSAY TO SCREEN FOR RESISTANCE TO INFECTION BY

PH Y T OPH T HORA CAPSICI

110

ABSTRACT

Cucumber fruit rot caused by Phytophthora capsici [Leonian] has become a
persistent threat, reducing harvestable yields and rendering fields unsuitable for
cucumber production. Identification and utilization of cucumber resistance to P. capsici
would provide a viable disease management strategy for producers. Therefore, the
objectives of this study were to develop a screen for testing detached cucumber fruit for
resistance to P. capsici and to screen cucumber culti gens for resistance. Four P. capsici
isolates (differing in their sensitivity to the fungicide mefenoxam and compatibility type)
were compared for pathogenicity in 1999 and 2000. No significant differences were
found among isolates. From 1999 to 2005, 432 cucumber cultigens (commercial cultivars
and plant introductions) were grown according to standard practices at Michigan State
University research farms in fields with no history of P. capsici. Commercially mature
fruit were harvested, inoculated with P. capsici, and rated for lesion diameter, sporulation
diameter, and density of sporulation. This is the first report using an unwounded fruit
screen to analyze cucumber resistance to P. capsici. Although no fruit exhibited

complete resistance to P. capsici, some cultigens showed limited pathogen sporulation.

111

INTRODUCTION

Fruit rot caused by the fungal-like oomycete pathogen, Phyophthora capsici
[Leonian] has become a limiting factor for cucumber producers in Michigan and has been
reported in several regions of the US. (6, 14). Phytophthora capsici has been found to
infect a wide range of Solanaceous and Cucurbitaceous hosts worldwide (4, 5, 7).
Cucumber fi'uit are especially susceptible. Fields of healthy-appearing vines have been
bypassed, and harvested loads rejected at the processor due to widespread fruit rot (6).
While the root and crown of the cucumber plant may become infected by P. capsici.
infection of these tissues is primarily limited to the seedling stage. Fruit may become
infected while in the field with the disease progressing during storage and transit;
symptoms and/or signs become evident after delivery to the processor or retailer (6).

Pathogen infection is most obvious as white powdery growth resulting from
sporangia produced in mass on the surface of infected cucumber fruit. In the presence of
free water, sporangia release 20 to 40 motile zoospores capable of causing widespread
plant infection in a short period of time (6). Oospores are over-wintering, long-term,
primary survival structures that form when the A1 and A2 compatibility types (CTS) of P.
capsici come together (2, 10). Both the A1 and A2 CTs were present in fields sampled in
Michigan, and in other vegetable-producing states (1, 6, 8, 17). Germinating oospores
infect the crop in the spring (11).

While crop rotation is the foundation of disease management, the long-term
survivability of oospores limits the effectiveness of this strategy for P. capsici (6, l 1, 17).
Historically, growers have relied on the phenylamide fungicide mefenoxam to manage

this disease, but resistant P. capsici isolates have been identified in Michigan, North

112

Carolina, and New Jersey (8, 9, 12, 14, 15). While other fungicides are available, they
provide varying levels of control, and the required frequent applications increase
production costs. In some cases, lengthy pre-harvest intervals limit fungicide use during
fruit development (6).

Whenever available, genetic resistance is a preferred disease management tool.
Frequently, screening for disease resistance is performed on seedlings because of space
and time considerations, as has been done for cucumber with respect to several fungal
pathogens (3). However, disease resistance in seedlings is not known to be correlated
with fruit resistance, and comparative susceptibility of cucumber vines and fruits in the
field suggests that more than one mechanism may be involved. In pepper, resistance to
P. capsici is under different genetic control in different tissue types (19, 20).

Since the primary impact of P. capsici in the field is on fruit, a methodology for
screening cucumber fruit for resistance to this pathogen is needed to evaluate cultigens.
Our research objectives included 1) developing an effective methodology for screening
cucumber fruit for resistance to P. capsici and 2) screening the fruit of C ucumis satit'us

P13 and commercial cultivars to identify resistance for breeding.

113

MATERIALS AND METHODS

Screening Methodology

Isolate maintenance and selection: Fresh cultures of each isolate were obtained
by transferring agar plugs from long-term stock cultures (stored at 20°C in sterile micro-
centrifuge tubes with l-ml of sterile water and a sterile hemp seed) onto V-8 juice agar
(16 g agar, 3 g CaC03, 160 ml unfiltered V8 juice, and 840 ml distilled water). Cultures
were maintained at room temperature under continuous fluorescent lighting. Seven-mm
diameter agar plugs from the margins of actively growing colonies were then transferred
to new V-8 juice agar and maintained under the conditions indicated above.

Phytophthora capsici isolates OP97, SP98, SFF 3, and SF3 were selected for
comparison of cucumber fruit disease response in 1999 and 2000. Isolate notation refers
to cultures maintained in the laboratory of Dr. Mary Hausbeck in the Department of Plant
Pathology at Michigan State University (MSU). All four isolates were collected in
Michigan from infected cucurbit crops. Isolates were characterized according to
compatibility type (CT) and sensitivity to mefenoxam as described by Lamour and
Hausbeck (8). Both OP97 (A1 CT) and SP98 (A2 CT) are fully sensitive to mefenoxam.
Isolate SFF3 is an A2 CT and is insensitive to mefenoxam. Isolate SF3 is an A1 CT with
intermediate sensitivity to mefenoxam. For each P. capsici isolate, disease was evaluated
on 66 cucumber cultigens in 1999, and on 58 cultigens in 2000 at three days post
inoculation (dpi). Disease parameters included sporulation density (0=none, 1=light,
2=moderate, 3=heavy sporulation), sporulation diameter (cm, white powdery sporulation,
Fig. 1D), and diameter of water-soaking (cm, visible dark discoloration on the fruit

surface, Fig. 1D). Data were subjected to analysis of variance using the PROC MIXED

114

 

 

procedure of SAS (SAS Institute Inc., Cary, NC) and means were compared using
Fisher’s Protected LSD test (P=0.05).

Inoculation evaluation: With the exception of the zoospore method (described
below), all inoculation tests were conducted with the following parameters. A 7-mm-
diameter plug of actively expanding P. capsici mycelia/sporangia was used to inoculate
commercially-mature fruit (2.54—5.08 cm diameter) (18) that were placed in aluminum
trays and covered with plastic wrap. A wetted paper towel was placed inside each
incubation chamber to provide 100% relative humidity (Fig. 1B). Trays were exposed to
constant overhead fluorescent lighting at 23-25 °C. Fruit were evaluated for disease 3 dpi
by measuring three parameters: water-soaked lesion diameter, sporulation diameter, and
sporulation density. In the first test, fruit were not wounded, and a sterile, plastic micro-
centrifuge tube (with the cap removed) was placed over the agar plug and fixed to the
fruit with petroleum jelly to maintain high humidity during initial infection (Fig. 1C). In
a second test, fi'uit were wounded by penetrating the fruit surface with a sterile syringe
needle at the site of inoculation. Plastic micro-centrifuge tubes were placed over the
inoculum plug and sealed to the fruit surface as indicated in the non-wounded fruit test.

Placing micro-centrifuge tubes over mycelial/sporangial plugs was compared with
inoculation without tubes in a third test. Unwounded fruit were inoculated, as described
above, however sterile micro-centrifuge tubes were not placed over the
mycelial/sporangial plugs during incubation. All other experimental conditions and
disease evaluations were carried out as previously described.

Zoospore inoculum was prepared by placing a 7-mm-diameter plug of a 7-day-

old culture in a sterile 1.5-ml plastic micro-centrifuge tube with l-ml of sterile, de-

115

 

Figure l. Cucumber fi-uit production and inoculation technique. A) Field production

of cucumber fi'uit (10 days post planting) at Plant Pathology Farm, Michigan State
University. B) Inoculation chamber for screening cucumber fruit for resistance to P.
capsici. C) Inoculated cucumber fruit 3 days post inoculation (dpi). D) Close up of
inoculated cucumber fruit 3 dpi (micro-centrifuge tubes removed for disease
evaluation). Note water-soaking (ws) and pathogen sporulation (s). (Images in this

dissertation are presented in color).

116

 

ionized water. The mixture was incubated at 4 °C for 20-min, followed by a 20-min
incubation at 23-25 °C to induce zoospore release. A 50-ul droplet of zoospore
suspension was applied to the surface of each unwounded fruit and covered with a sterile
1.5 ml micro-centrifuge tube (with the cap removed), and fixed to the fruit with
petroleum jelly. All other experimental conditions and disease evaluations were carried
out as described above. Each of the inoculation tests were conducted on 4 fruit per
cultigen for each of 5 cultigens and were repeated four times.
Germplasm Screening

Cucurbit germplasm accessions (Plant Introductions or PIs) were provided by the
United States Department of Agriculture Agricultural Research Service National Plant
Germplasm System (USDA-ARS-NPGS), North Central Regional Plant Introduction
Station (N CRPIS), Ames, Iowa. Cucumis sativus germplasm was chosen with
phenotypic characteristics similar to those of commercial pickling cucumbers (i.e.
‘Vlaspik’). In addition, newly-released commercial cucumber hybrids were also screened
(Appendix A.1). Germplasm descriptions can be found at httQ://www.ars-grin.gov/nggs.
A total of 432 cultigens were screened during 1999 to 2005 (Table 1, Appendix A. 1 ).
The pickling cucumber ‘Vlaspik’ was grown as a commercial standard in 1999 to 2005.

Cucumbers were grown according to standard management practices in fields
with no history of P. capsici. In early June, cucumbers were direct-seeded in rows that
were 61.0 cm apart with an in-row plant spacing of 30.5 cm. Rows were bedded and
covered in black polyethylene mulch with sub-mulch drip irrigation for weed control and
overall plant health maintenance (Fig. 1A). Pest control and fertilization were applied

according to standard commercial practices. Commercially mature (5.1 to 7.6 cm

117

Table 1. Cucumber culti gens screened for fruit resistance to Phytophthora capsici.

 

 

 

 

 

Year Cultigen
Total # of # Plant # Cultivated # Selected for
cultigens Introductions varieties re-evaluation
screened

1999 78 38 40 18

2000 55 30 25 1 1

2001 42 13 29 8

2002 60 41 19 10

2003 52 33 19 12

2004 107 77 30 38

2005 38 18 20 0

Total 432 250 182 97

118

diameter) fruit were harvested, subjected to a 5-minute immersion in a 5% sodium
hypochlorite solution, then rinsed in distilled water. Fruit were allowed to dry under
ambient conditions and labeled according to cultigen. Fruit were harvested from the field
on two or more separate occasions afler observation of the first commercially mature
fruit. Two to ten fruits were collected for each harvest and were inoculated by placing
plugs of mycelial/sporangial inoculum on unwounded fruit. Plugs were covered with
sterile micro-centrifuge tubes and fixed to fruit surfaces with petroleum jelly (Fig. 1C).
Fruit were incubated in aluminum trays covered with plastic wrap under constant
fluorescent lighting at 23-25 °C (Fig. 1B). Disease was evaluated by measuring the
water-soaked lesion diameter, sporulation diameter, and sporulation density 3 dpi (Fig.
1D). In all tests, four to eight fruit per culti gen were tested for each of four harvests.
Cultigens whose fruit exhibited limited lesion development and pathogen
sporulation were re-screened in subsequent years. Criteria for re-screening included a
sporulation density and sporulation diameter of $1 .0 cm. In some years, lesion diameter
was also taken into consideration. F orty-five cultigens were screened in 2 or more years
(Table 2). Thirty-one (69%) of the cultigens selected for re-screening were PIs; the
remaining 14 were commercial cultivars (Table 2). Of the 25 cultigens selected for re-
screening in 1999, 20 (80%) were P15 and only 5 were commercial cultivars (Table 2). In
2000, a total of 29 cultigens were selected; 20 were P15 (69%) and 9 were commercial
cultivars. In 2001-2003, 12 to 16 cultigens were re-screened; more than half of these
culti gens were PIs. Sixty-seven percent of the 12 cultigens selected for re-screening in

2004 were commercial cultivars (Table 2).

119

 

 

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121

RESULTS

Screening Methodology

The mycelial/sporangial plug inoculation method using micro-centrifuge caps was
an effective screening method for establishing P. capsici infection on susceptible
cucumber fruits (Fig 1B, C). Wounding was not necessary for successful infection.
Covering the mycelial/sporangial plugs with sterile micro-centrifuge tubes was preferred,
as uncovered plugs rapidly desiccated and disease did not progress. Zoospore
inoculations yielded fruit symptoms similar to those observed with mycelial plugs,
however, with less consistency, since zoospores require water. Applying uniformly-sized
droplets on the hydrophobic surface of the cucumber fruit was difficult.

The four P. capsici isolates representing different CT and mefenoxam sensitivity
did not differ significantly for any of the three disease parameters (sporulation diameter,
sporulation density, and lesion diameter) (Fig. 2). All four isolates induced sporulating
lesions of approximately 2.75 cm in diameter and sporulation density of 1.5 at 3 dpi;
similar results were observed for lesion diameter (all approximately 6.0 cm) (Fig. 2).
Therefore, a single isolate, OP97, was selected to carry out all subsequent culti gen
inoculations.
Germplasm Screening

Of the 432 cultigens evaluated, 250 were P15 and 182 were commercial cultivars.
No cultigens exhibited complete fruit resistance to P. capsici infection. However, 1 % of
the PIs and 8% of the commercial cultivars exhibited limited sporulation (e. g.,

sporulation diameters less than 2.0 cm or sporulation densities less than 1.0) (Fig 3A, B

122

 

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(scale 0-3)

 

Sporulation Diameter
(cm)

 

Lesion Diameter
(cm)

 

 

 

OP97 SP98 SFF3 SF3

Phytophthora cquici Isolates

Figure 2. Comparison of disease ratings (sporulation density, sporulation diameter, and
lesion diameter) of 4 P. capsici isolates (OP97, SP98, SFF3, and SF3) on cucumber fruit
during 1999 and 2000. No significant differences were found between isolates using

Fisher’s Protected LSD test (P=0.05).

123

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and Table 2). More than half of the cultigens had water-soaked lesions of less than 3.0
cm (Fig. 3C). Promising cultigens that were re-screened either failed to again exhibit
limited sporulation and were abandoned, or exhibited limited sporulation and were
further evaluated in subsequent years. All cultigens that were screened in more than two
years performed consistently with limited sporulation. Some of the least susceptible
included the commercially available cultivars Discover, Excel, Vlaspik, and Vlasspear
(Table 2). No PIs were reproducibly less susceptible than ‘Vlaspik’ when retested in
multiple years.

DISCUSSION

As P. capsici becomes more prevalent on vegetable crops in the US. (6), it would
be highly valuable to identify host plant resistance to the pathogen for incorporation into
a disease management strategy. For cucumber crops, fruit resistance is crucial because
fruit, which are the marketable portion of the plant, also appear to be the most susceptible
part of the plant. Thus, an essential first step is the establishment of screening procedures
specifically for cucumber fruit.

Our screening methodology included unwounded cucumber fruit inoculated with
mycelial/sporangial plugs of P. capsici covered by micro-centrifuge tubes and incubated
at high humidity for three days. This screening method, which was efficient and
reproducible, does not require wounding which may interfere with the host-patho gen
interaction and result in fruit susceptibility different than that expected under field
conditions. In the field, fruit infection occurs after irrigation or driving rain with the
splash dispersal of mycelial fragments, sporangia, and zoospores (16). The use of

mycelial/sporangial plugs as inocula allowed for more consistent infection than zoospore

125

inoculum due to the difficulty in quantifying the number of zoospores in each droplet and
assuring that each droplet remained intact on the fruit surface for three days.

No significant differences in disease reactions were determined among the four P.
capsici isolates of differing mefenoxam sensitivity or compatibility type. Therefore, a
single P. capsici isolate, OP97, was used for fruit inoculations. Isolate OP97 maintained
consistent growth and virulence after repeated sub-culturing, and has also been used
extensively in previous studies as a robust Al CT standard (8). Using a single isolate
simplified our screening. It is important before initiating a screen, however, to verify the
virulence of the selected P. capsici isolate on susceptible host tissue.

Over a 6-year period, we evaluated the commercially-mature fruit of 432
cucumber cultigens for resistance to P. capsici and found that none exhibited complete
resistance to pathogen infection. Some culti gens, including several commercial cultivars,
however, showed limited pathogen sporulation on fiuit. None of the P15 performed
consistently with limited pathogen sporulation, as did the best cultivars. Until a resistant
source is developed, using cultivars that limit pathogen sporulation may be beneficial in
managing field disease.

Overall disease on cucumber fruit in 2004 was less than that observed during
1999 to 2003 and 2005 making it difficult to differentiate among genotypes; therefore
2004 results were not included in the data summaries. The reduced disease development
may be due to weather and/or culti gen selection. The average air temperature during the
cucumber-growing season at the Plant Pathology Farm, MSU, was 25.8°C (80°F) for

1999-2003 and 2005, with 19.8 growing degree days wase 50), but only 23.0°C (75°F)

126

with 16 growing degree days in 2004. Cooler temperatures affect physiological
development of cucumber fruit, which may have impacted sensitivity to fruit rot (13).

In summary, we developed a direct cucumber fruit assay to screen for resistance
to infection by P. capsici. Screening of approximately 400 culti gens did not identify a
source of resistance superior to the partial resistance already present within many
commercial cultivars.

ACKNOWLEDGEMENTS

Dr. Kurt Lamour contributed significantly to the efforts of this work by initiating the
screening development in 1999 and 2000. I would like to acknowledge the Pickle and
Pepper Research Committee of Michigan State University (Pickle Packers International,
Inc.), the Pickle Seed Research Fund (Pickle Packers International, Inc), Project
GREEEN (a cooperative effort by plant-based commodities and businesses with
Michigan State University Extension, the Michigan State University Extension, the
Michigan Agricultural Experiment Station, and the Michigan Department of Agriculture),
Michigan Department of Agriculture Specialty Crop Block Grant, and Michigan
Agriculture Experiment Station. Thanks also to M. Bour and K. Cervantes for technical

assistance and B. Cortright for field assistance.

127

10.

11.

12.

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APPENDIX

130

 

Table A. l. Cucumis sativus cultigens screened for fruit resistance to P. capsici during 1999 to 2005.

 

 

 

Cultigen, Provider Year(s) Cultigen, Provider Year(s)
screened screened

ACX 18, Abbott & Cobb5r 99 Bush Baby l4lH, Liberty/StokesP 03
ACX 5001, Abbott & CobbS 99 Bush Hyb 141G, Liberty/StokesP 03
ACX 5002, Abbott & Cobbs 99 Caipira Hyb Ag-221, SeminisS 04,05
Ames 13247, USDAS 04 Calypso, Atlas SeedsP 99
Ames 13333, USDA 02,03 Campbell 4177, SeminisP 03-05
Ames 1760, USDAS 02 Carolina, Atlas SeedsP 99
Ames 20089, USDA O4 Colt Hyb, AsgrowP 00
Ames 21224, USDAP 04 Colt, SeminisP 02
Ames 23009, USDAS 04 Cool Breeze, Harris Moran" 04
Ames 3941, USDAS 02 Country Fair Hybrid, Park'sP 05
Ames 3942, USDAS 02 Cross Country, Harris MoranP 99,04,05
Ames 3943, USDAS 02 Cyclone, AsgrowS 00
Ames 3944, USDA" 02,03,04 Dasher II, PctosecdS 99
Ames 3945, USDA” 02 Daytona 149 o, Liberty/StokesP 03
Ames 3946, USDAP 02 Discover M Hybrid, AsgrowP 99,01
Ames 3947, USDA 02 Discover, SerninisP 00,02
Ames 3948, USDA 02 Diva, Harris MoranS 04,05
Ames 3949, USDA 02 Eclipse 146E, Liberty/StokesP 03
Ames 3950, USDA 02,03 Eureka, SiegersP 00
Ames 3951, USDA 02 EX 1911 155633, SeminisP 99
Ames 4421, USDA 02 EX 1914 183491, SerninisP 99
Ames 4759, USDA 02 Excel M, Asgrow” 99,01
Ames 4832, USDA 02 Excel, SerninisP 00,02
Ames 4833, USDA 02 Fancipak, Asgrow, Seed WayP 99,04
Ames 7118, USDA" 99 Fanfare 127G, Liberty/StokesP 03-05
Ames 7730, USDA 02 Feisty, Harris MoranP 04-05
Ames 7731, USDA 02 FMX 5020 F1, Harris MoranP 99
Ames 7735, USDA 02 General Lee, Harris Moran8 99
Ames 7736, USDA 02 Ginga Hyb Ag-77, SeminisS 04
Ames 7737, USDA 02 Green Slam, Siegers 04
Ames 7738, USDA 04,05 Greensleeves, Harris MoranS 99
Ames 7739, USDA 02 Gy4, NCSU 02
Ames 7740, USDA 02 HMX 3469 F1, Harris MoranP 99
Ames 7738, USDA 04,05 HMX 8460 F1, Harris MoranP 99
Ames 7739, USDA 02 HMX 8461 F1, Harris MoranP 99
Ames 7740, USDA 02 Impact, SiegerS 04
Ames 7741, USDA 02 Indio 149F, Liberty/Stokes" 03
Ames 7742, USDA 02 Indy, Sieger/Harris/Seedways 04
Ames 7744, USDA 02,03 Intimidator, Seed Ways 03
Ames 7742, USDA 02 Jackson, Sun Seeds" 99
Ames 7744, USDA 02,03 Jade, Harris Moran" 03
Ames 7745, USDA 02 Johnston, NCSU 02
Ames 7749, USDA 02 Lafayette Classic, Sun SeedsP 99,00
Ames 7750, USDA 02 Lucia, NCSU 02
Ames 7751, USDA 02 M17, NCSU 02
Ames 7752, USDA 02 Magic Hyb, SeminisP 03
Ames 7753, USDA 02 Manteo, NCSU 02
Ames 7785, USDA 02 Marketmore 76, Hollar SeedsP 03
Arabian Hybrid, AsgrowP 00,0l Meteor, AsgrowS 00
Arabian, SerninisP 02 NC-43, NCSU 02
Aflraph, Seed WayS 04,05 Niagra 144W, Liberty/StokesP 03

131

Table A. 1. Continued.

 

Cultigen, Provider

Orient Express, Harris Morans
Palomino Hyb, AsgrowP

Palomino, Seminis

Panther, Sun SeedsS
Patio Pickle, Harris MoranP
Patton 9228, ScedWay"

PI 103049, USDAS
PI 109482, USDAs
PI 109484, USDA
PI 114339, USDAS
PI 137850, USDAs
Pl 163213, USDA
PI 163214, USDA
P1 167223, USDA
PI 169304, USDAS
PI 172838, USDAS
PI 183056, USDA
PI 183127, USDA
PI 183224, USDAS
PI 197085, USDA
PI 197086, USDA
PI 197087, USDA
Pl 197088, USDA
PI 206043, USDAs
PI 209064, USDA
PI 209065, USDAS
PI 209067, USDA
PI 209068, USDA
PI 209069, USDA
PI 211979, USDA
PI 211980, USDA
PI 220860, USDAs
PI 226461, USDAS
PI 227209, USDA
PI 227210, USDAS
PI 234517, USDA
PI 249561, USDA
PI 249562, USDA
Pl 257486, USDA"
PI 262990, USDA"
PI 264664, USDA"
PI 264667, USDAS
PI 267741, USDAS
PI 267935, USDAS
PI 267942, USDA
PI 271326, USDA
Pl 271327, USDA
PI 271328, USDA
PI 271753, USDA"
PI 271754, USDA"
PI 279466, USDA

Year(s)
screened
04

00

02

99
04,05
03

04

04

04

04
04,05
99,00
00

99
04,05
04

02

02

04

01 ,02
99

00
99-02
04,05
99

04

00

00
99,00
99,00
00

04

04
99,00
00

99
00,01
99-02
03,04
04

04

04

04

04

99

01 ,02
00-02
99,00,03
04
04,05
99,00

Cultigen, Provider

P1279467, USDA
PI 279468, USDA
PI 283899, USDA"
P1288238, USDA
P1292012, USDAS
P1302443, USDAS
PI 306179, USDAS
PI 306180, USDA
PI 306785, USDAS
PI 314425, USDA
Pl 321006, USDA
PI 321007, USDA
PI 321008, USDA
PI 321009, USDA
PI 330628, USDA
PI 342950, USDA"
P1354952, USDA"
PI 358813, USDA
PI 358814, USDA
PI 360939, USDA"
P1364472, USDAS
PI 372893, USDAS
PI 374694, USDAS
PI 390239, USDA
PI 390240, USDA
PI 390241, USDA
PI 390244, USDA
P1390246, USDA
PI 390251, USDAS
PI 390252, USDAS
P1390253, USDA"
PI 390261, USDA
PI 390262, USDA
PI 390263, USDA
PI 390266, USDAS
PI 390529, USDA
PI 391570, USDA
P1401732, USDA
P1401733, USDA
P1414157, USDAS
P1414158, USDAS
P1414159, USDAS
P1414716, USDAS
P1 418963, USDAS
P1418964, USDA
P1419009, USDAS
P1420150, USDA"
P1422167, USDAS
P1422168, USDA"
P1422169, USDA"
P1422170, USDA"

132

Year(s)
screened
99,00
99,00
04

99

04

04

04

04

04
04,05
01

01
99-01 ,03
00
99,00
03

03
99,00
00

03
04,05
04,05
04

00
99,00
99

99

99

04

04

04

01
99,01
00

04

99
99,03
01

01

03
04,05
04

04

04

99
04,05
04
04,05
04

04

03

Table A. 1. Continued.

 

Cultigen, Provider

PI 422171, USDA"
P1422172, USDA"
P1 422173, USDA"
PI 422174, USDA"
PI 422176, USDA"
P1422179, USDA"
PI 422180, USDA"
P1422181, USDA"
PI 422182, USDA

PI 422183, USDA"
P1422185, USDA"
P1422186, USDA"
P1422188, USDA"
PI 422189, USDA"
PI 422190, USDA"
P1422191, USDA"
P1422198, USDA"
P1 422199, USDA"
PI 422200, USDA

P1426169, USDA

P1426170, USDA

P1 432851, USDA

P1432855, USDA

PI 432865, USDA

PI 432867, USDA

P1432868, USDA

P1432890, USDA

PI 451975, USDA"
PI 451976, USDA"
PI 458852, USDA"
PI 466922, USDA

P1 483339, USDA

P1490996, USDA"
P1 500359, USDA"
P1 504559, USDA"
PI 504563, USDA"
PI 504569, USDA"
Pl 504813, USDA"
Pl 508453, USDA"
PI 508455, USDA"
PI 508456, USDA"
P1 508458, USDA"
PI 508459, USDA"
PI 508460, USDA"
PI 511818, USDA"
Pl 511819, USDA"
P1 512336, USDA"
Pl 512638, USDA"
P1 606000, USDA"
P1 606032, USDA"
PI 606058, USDA"

Year(s)
screened
03

03

03

03

03

03
03,04
04,05
99,03
03

03

03

03

03

03

03

03

03

04
99,00
99,00
99

99

99
99,00
00

99

04

04

04

99

99

04

04
04,05
04

04

04

03

04

03

04

04

O4

04
04,05
04

04

()4
04,05
04

Cultigen, Provider

PI 606066, USDA"
PI 618860, USDA"
PI 618861, USDA"
Pl 618862, USDA"
Pl 92806, USDA"
Pioneer, Atlas SeedsP
Prancer, SiegerS

Premier Hyb, SeminisS
Raider, Harris MoranS

Raleigh, NCSU

Regal F1, Harris MoranP
Reisenschal B1 SMP, Vlasic"
Reisenschal BZ SMP+, VlasicP
Reisenschal B3 SMPE, Vlasic"
Reisenschal B4 SMPE+, VlasicP

Reisenschal, VlasicP

Royal F1, Harris MoranP
Salad Bush, Harris MoranS
Salty 142C, Liberty/Stokes"

Sassy, SeedWay
Shelby, NCSU

spear It 141E, Liberty/Stoke"

Speedway, PetoseedS

Spunky, Harris MoranP

SRQP 2391, Sun Seeds"
SRQS 2387, Sun Seeds"
SRQS 2389, Sun Seeds"

SS-58137, Sun Seeds
SS-58139, Sun Seeds
SS-58141, Sun Seeds
SS-58142, Sun Seeds
SS-58143, Sun Seeds
SS-58144, Sun Seeds
SS-58145, Sun Seeds
SS—58146, Sun Seeds
SS-58147, Sun Seeds
SS-58148, Sun Seeds
SS-58149, Sun Seeds
SS-58150, Sun Seeds
SS-58151, Sun Seeds
SS-58152, Sun Seeds
SS-58153, Sun Seeds
SS-58154, Sun Seeds
SS-58155, Sun Seeds
SS-58456, Sun Seeds

Stallion 193782, Seminis"
Stallion Hyb, AsgrowP

Sumter, Atlas SeedsP

SVR04504229, Seminis"
SVR04506116, Seminis
Sweet Slice, Seed WayS

133

Year(s)
screened
04,05
04
03-05
04,05
04

99
04,05
04
04,05
02

99

00

00
00

00

00

99

04

03

05

02

03

99
04,05
99

99

99

01

01

01

01

01

01

01

01

01

Ol

01

01

01

01

01

01

01

01

99
00,01
99

02

02

04

Table A. 1. Continued.

 

C ultigen, Provider

Year(s)
screened

 

Sweet Success, Harris MoranS
sx 23 87CW/Lynx, Seed Way"
sxo 3534. Sieger"

Tamor Hyb, AsgrowP

Tasty Green, Seed WayS
Thunder, Asgrow/SiegerS
Thunderbird, Seed Way"

Transamerica F 1, Harris MoranP

Transamerica, Sun SeedsP
Turbo, Harris/Stokess
Ultra Pak, Stokes"
Victoria, Sun SeedsP
Vlaspick, VlasicP

Vlaspik Bl SMP, VlasicP
Vlaspik B2 SMP+, VlasicP
Vlaspik B3 SMPE,v1asic"
Vlaspik B4 SMPE+, Vlasic"
Vlaspik VGA733, Seminis"
Vlaspik, AsgrowP

Vlaspik, SeminisP
Vlaspik, VlasicP
Vlaspik+M Hyb, AsgrowP
Vlasset, AsgrowP

Vlasset, Seminis"
Vlasspear Hyb, As rowP
Vlasspear, Seminis
Vlasstar B, AsgrowP

WI 1983 G, 1997

WI 5207, 2000

WI 5551, 1994

WI 6632 E, 1997

WI GY 14, 1998

WI SMR 18, 2000
Wisconsin, Atlas SeedsP
XP 1904, Seminis"
Zapata, Seed WayP

04,05
04,05
04,05
99

04
00,04,05
03,04
99

00
04,05
99

99

99

00

00
00,03-05
00

99

00

02

00
00,01
99

02

99
00,02-05
99

Ol

01 '
01

0 l

01

01

99

02

03

P Indicates that the cultigen exhibits fruit of a pickling cucumber type.
SIndicates that the cultigen exhibits fruit of a slicing cucumber type.

134

   

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