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This is to certify that the
dissertation entitled

EVALUATION OF THE ROLE OF PLANT ARCHITECTURE
AND CUCUMBER FRUIT DEVELOPMENT IN
PHYTOPHTHORA CAPS/CI DISEASE DEVELOPMENT

presented by

KAORI ANDO

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5/08 K’lProj/AccaiPres/CIRC/DateDue.indd

EVALUATION OF THE ROLE OF PLANT ARCHITECTURE AND CUCUMBER
FRUIT DEVELOPMENT IN PHYTOPHTHORA CAPSICI DISEASE DEVELOPMENT

By

Kaori Ando

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Plant Breeding & Genetics — Horticulture

2009

ABSTRACT

EVALUATION OF THE ROLE OF PLANT ARCHITECTURE AND CUCUMBER
FRUIT DEVELOPMENT IN PHYTOPHTHORA CAPSICI DISEASE DEVELOPMENT

By
Kaori Ando

Fruit rot caused by Phytophthora capsici Leonian is an increasingly serious
disease affecting production of cucumber (C ucumis sativus L.) and other cucurbit crops
in many parts of the US. The absence of genetically resistant cultivars and rapid
development of fungicide resistance makes it imperative to develop integrated disease
management strategies. Cucumber fruit which come in direct contact with the soil-borne
pathogen are usually located under the canopy where moist and warm conditions favor
disease development. Screening a collection of 150 Plant Introductions (PI) revealed
variation for an array of architectural traits. One of the compact lines (PI 308916), which
had a tendency to hold young fruit off the ground, exhibited lower disease occurrence
which was not due to genetic resistance, suggesting that architecture which allows less
contact of fruit with the soil could be useful for P. capsici control for cucumber. In the
course of screening for resistance sources among cucumber PIs, fruit age/size was shown
to be a factor in susceptibility to infection by P. capsici. Inoculation of greenhouse-grown
fruits of known ages showed that cucumber fruits were most susceptible to P. capsici
when they were very young and rapidly elongating, but then developed age-related
resistance (ARR) as they approach full length at 10-12 days post pollination (DPP). This
was observed in both the greenhouse and ﬁeld and for several genotypes. Testing of
seven additional cucurbit crops, zucchini, summer squash, acorn squash, pumpkin,

butternut squash, melon, and watermelon also showed ARR, but to varying degrees. In

the ﬁeld, infection primarily occurs at the blossom end. Inoculation of cucurbit fruits of
various ages with P. capsici at the peduncle and blossom ends showed that as fruit age
increased, the peduncle end became less susceptible sooner than the blossom end,
suggesting a developmental gradient within the fruit inﬂuencing susceptibility for
cucumber, zucchini, acorn, and butternut squash fruits. To understand the basis for ARR
in the cucumber ﬁ'uit-P. capsici interaction, and to increase understanding of early fruit
development in cucumber, morphological and global gene expression analyses were
performed. Morphological changes associated with cucumber ﬁ'uit development were
catalogued for hand pollinated greenhouse grown fruits at 0, 4, 8, 12, 16, 20, 26, and 32
DPP. These fruits were also collected to generate cDNA libraries for 454 pyrosequencing
technology developed by Roche®. Young cucumber fruits show marked changes in fruit
size, cell size, surface wax, chlorophyll content and patterns, wart formation, spine
development, and placenta and seed development. 454 pyrosequencing analyses yielded
187,406 clean reads, of which 88 % could be assembled into 13,879 contigs. The number
of sequences per contig, which is reﬂective of transcript abundance, ranged from 2-5,167.
BLAST analysis of the most highly represented transcripts against the nr protein
sequence NCBI database, indicated high representation of genes associated with protein
synthesis, ﬂowers, fruits or seeds of other species, latex related proteins, lipid
biosynthesis, cell expansion, defense, phloem transport, and photosynthesis. Results of
454 and qRT-PCR for selected genes were comparable, indicating that the 454
transcriptome sequencing can be used for analyzing relative gene expression during fruit
development. These information will contribute to the understanding of biology of

cucumber ﬁ'uit development and possible relationship to age-related resistance.

ACKNOWLEDGEMENTS

I would like to thank all my committee members, Drs. Mary Hausbeck, Ray
Hammerschmidt, Jim Kelly, Mathieu Ngouajio, and Rebecca Grumet for their support
and guidance. I want to show special appreciation to Rebecca for her patience, coaching,
and providing me a great opportunity to pursuit my dream.

Great thanks to past and present members of Grumet Lab. I especially thank Sue
Hammar and Holly Little for their friendship, moral support, and making my life both
inside and outside of the work very stimulating. I would like to thank Zhulong Chan, a
molecular technique sensei, for giving me advice. Thanks to all the undergraduates and
highschool students who helped me throughout the years, especially Dan Raba who made
weeding enjoyable and entertaining.

I appreciate all of fellow “joint labbers” for their helpful suggestions and
discussions. I would like to acknowledge Dr. lBukovac for giving me an opportunity to
work as a research assistant in his lab and for greatly inspiring me to pursue higher
education. Also, I would like to thank Paolo Sabbatini for being such a fun and great
friend to me. I owe special thanks to Neil Adhikari for always being there for me and for
his understanding.

I would like to show gratitude to my wonderﬁll family for being there for me
despite me being more than 6,000 miles away. I love you all and am glad I have you in
my life.

It’s all worthwhile!

iv

TABLE OF CONTENTS

LIST OF TABLES .. ......................................................................................................... vi

LIST OF FIGURES ........................................................................................................... vii

CHAPTER 1: Literature Review
Introduction. . . .......................................................................................................... 1
Phytophthora capsici biology.. ............................................................................... 1
Phytophthora capsici control in cucumber production ............................................ 5
Genetic modiﬁcation of plant architecture for disease control ................................ 6
Age-related resistance ............................................................................................ 10
Genomic approaches to study fruit development in cucumber .............................. 13
Objectives of dissertation... ................................................................................... 15
Literature cited... ................................................................................................... 17

CHAPTER 2: Evaluation of altered cucumber plant architecture as an approach to reduce

Phytophthora fruit rot
Abstract .................................................................................................................. 25
Introduction ............................................................................................................ 26
Materials and Methods ........................................................................................... 31
Results .................................................................................................................... 35
Discussion .............................................................................................................. 50
Acknowledgements ................................................................................................ 53
Literature cited ....................................................................................................... 54

CHAPTER 3: Age related resistance in cucumber and various cucurbit fruit to infection
by Phytophthora capsici.

Abstract .................................................................................................................. 57
Introduction ............................................................................................................ 58
Materials and Methods ........................................................................................... 61
Results .................................................................................................................... 67
Discussion .............................................................................................................. 90
Literature cited ....................................................................................................... 96

CHAPTER 4: Cucumber ﬁ'uit transcriptome analysis by 454 sequencing

Abstract .................................................................................................................. 99
Introduction .......................................................................................................... 100
Materials and Methods ......................................................................................... 103
Results .................................................................................................................. 108
Discussion ............................................................................................................ 128
Literature cited ..................................................................................................... 136
CONCLUSIONS AND FUTURE WORK ...................................................................... 142

LIST OF TABLE

Table 2-1. Temperature under the canopy at cucumber fruit positions and cucumber fruit
weight, number of fruit, and disease occurrence for narrow, wide, and trellis plots
at 0 day post harvest (DPH) and 4 DPH ................................................................ 36

Table 2-2. Percent Phytophthora capsici-infected cucumber fruit rated at 4 days
postharvest and averaged over three harvest dates ................................................ 41

Table 2-3. Correlation (R2 value) between stern length, intemode length, leaf length and
width, and number of branches .............................................................................. 44

Table 2-4. Origin, architectural type, number of branches, main stem length, fruit
position, percent of infected fruit at O and 4 DPH, and susceptibility of selected
Plant Introductions for architectural effect on controlling Phytophthora capsici.45

Table 3-1. Age, size, and response to inoculation by Phytophthora capsici of greenhouse
grown cucurbit ﬁ'uits at various stages of development as determine by visual

properties ................................................................................................................ 87
Table 3-2. Symptom development on peduncle and blossom ends of cucurbit crops in

response to infection by Phytophthora capsici at 4 days post inoculation ............ 89
Table 4-1. Overview of 454 sequencing results from library construction 1 .................. 115
Table 4-2. Summary of 454 sequencing results for 8 days post pollination (DPP)

cucumber fruit transcript samples ........................................................................ 116
Table 4-3. Overall summary of contig assembly from combined 454 runs .................... 1 19

Table 4-4. Predicted function (based on BLAST analysis) and number of reads for highly
expressed cucumber fruit genes (2 0.15% transcript frequency) ......................... 121

Table 4-5. Presence/absence of the top 0.2 % cucumber fruit transcripts in the

International Cucurbit Genomic Initiative (ICuGI) EST libraries of cucumber fruit,
leaf, male and female ﬂower buds, melon fruit, and watermelon fruit ................ 124

vi

LIST OF FIGURES

Figure 2-1. Mean fruit yield per plot (4.6 m x 3 m)and percent disease occurrence at
harvest and four days post harvest at four harvest dates for standard, narrow, and
trellis plots .............................................................................................................. 37

Figure 2-2. Mean fruit number per plot (1.8 m x 3 m), percent disease occurrence at
harvest and four days post harvest for ‘Vlaspik’, determinate (dedeLL and dedell),
little leaf (DeDell, dedell, and H-19), and compact architectural types ................ 40

Figure 2-3. Frequency distribution of ﬁfty cucumber PIs showing architectural variation
relative to ‘Vlaspik’ for stem length, intemode length, number of branches, and
leaf width ............................................................................................................... 43

Figure 2-4. Mean fruit number per plot (1.8 m x 3 m) and percent disease occurrence at
harvest and four days post harvest for ‘Vlaspik’; mean of the commercial lines
‘Arabian’, ‘Colt’, ‘Palamino’, and ‘Stallion’; and reduced branching types (mean
of PI 192940, 227207, and 401734) ....................................................................... 48

Figure 2-5. Fruit position for PI 308916 early and late in the season ............................... 49

Figure 3-1. Frequency distribution showing number of cucumber genotypes for which
sampled fruit showed the indicated sporulation at 5 and 10 days post
inoculation .............................................................................................................. 68

Figure 3-2. Relationship between cucumber fruit age and size, and response to
inoculation with Phytophthora capsici .................................................................. 70

Figure 3-3. Mean disease rating at 4 days post inoculation of parthenocarpic and ﬁeld-
grown non-parthenocarpic cucumber fruit in relation to fruit length to inoculation
by Phytophthora capsici ........................................................................................ 73

Figure 34. Mean disease rating of 8 and 15 days post pollination exocarp section and
intact fruit underneath to infection by Phytophthora capsici zoospore
suspension .............................................................................................................. 74

Figure 3-5. Mean disease rating of 8 and 16 days post pollination to infection by
Phytophthora capsici zoospore suspension at 6 days post inoculation ................. 76

Figure 3-6. Mycelium inoculation of the peduncle and blossom ends of 6 to 14 days post
pollination fruit ...................................................................................................... 77

Figure 3-7. Response of different cucurbit fruit to inoculation with Phytophthora
capsici. ................................................................................................................... 80

vii

Figure 3-8. Fruit size of ﬁeld-grown cucurbit fruit vs. response to inoculation by
Phytophthora capsici at 4 days post inoculation ................................................... 82

Figure 3-9. Relationship between fruit age and size and response to inoculation by
Phytophthora capsici at 4 days post inoculation for greenhouse-grown fruit ....... 85

Figure 4-1. Protocol for preparation of cucumber fruit cDNA libraries for 454

pyrosequence analysis .......................................................................................... 107
Figure 4-2. Cucumber fruit cell size in relation to fruit age ........................................... 109
Figure 4-3. Surface property changes in early cucumber fruit development .................. 111
Figure 4—4. Total chlorophyll content in relation to fruit age ......................................... 112
Figure 4-5. Greenhouse grown cucumber fruit growth ................................................... 113

Figure 4-6. Pairwise scatter plot and regression analysis for 8 d-A and 8 d-B samples
expressed in transcripts per thousand .................................................................. l 18

Figure 4-7. Predicted gene ontrogy (GO) classiﬁcation from BLAST analysis of
transcript represented at frequency of > 0.1% against TAIR 7.0 database .......... 120

Figure 4-8. Relative transcript levels of selected highly expressed genes in 20 vs. 4 days
post pollination fruit measured by 454 sequencing and quantitative PCR .......... 125

Figure 4-9. qRT-PCR analysis of the selected highly expressed genes in 4, 12, 20 and 32
days post pollination fruit .................................................................................... 127

Figure 4-10. External and internal development of cucumber fruit at O, 4, 8, 12, 16, 20,
26, and 32 days post pollination fruit ................................................................... 129

viii

Chapter 1

Literature Review

Introduction

Cucumber (C ucumis sativus L.) is cultivated in tropical and temperate regions of
the world, and its origin is thought to be Africa or Central/East Asia (Miller and Wehner,
1989). Cucumber fruits are consumed fresh or processed into pickles. Michigan is the
top processing (pickling) cucumber producing state in the United States. In 2008, 31,000
acres of pickling cucumber were planted in Michigan, with a gross value of 41 million
dollars (United States Department of Agriculture National Agricultural Statistics Service,
2008). The pickling cucumber production acreage has remained constant in recent years,
however, maintaining and increasing yield has become difﬁcult for growers due to
oomycete diseases including downy mildew caused by Pseudoperonospora cubensis and
fruit rot disease caused by Phytophthora capsici (Colucci et al., 2006; Hausbeck and

Lamour, 2004; Lamour and Hausbeck, 2000, 2001a,b, 2002).

Phytophthora capsici biology

Phytophthora capsici belongs to the kingdom Chromista and the class Oomycetes
(Agrios, 1997). Members of the genus Phytophthora, which means plant destroyer in
Greek, often cause disastrous diseases such as the potato leaf blight caused by
Phytophthora infestans that led to the Irish famine in the 1840s (Agrios, 1997; Erwin and
Ribiero, 1996; Judelson and Blanco, 2005). Phytophthora capsici was ﬁrst described as a

host speciﬁc pathogen of chili pepper (Capsicum annum L.) occurring in New Mexico

(Leonian, 1922). Since then, many crops including woody plants, omamentals, and
vegetables have been reported to be hosts (Erwin and Ribeiro, 1996). Although
incidence of cucumber infection was ﬁrst reported in Colorado in 1936 (Kreutzer, 193 7),
it has only recently become a common problem in cucumber production areas (Babadoost,
2004; Hausbeck and Lamour, 2004). Once cucumber ﬁelds are infected by Phytophthora
capsici, severe yield loss due to infection of the fruit is frequently observed (Babadoost,
2004; Hausbeck and Lamour, 2004). Phytophthora capsici infected cucumber fruit
typically develop a slightly discolored and depressed fruit surface which looks like water
soaking, subsequently white cottony mycelium cover the infected area. Mycelium
produce sporangiophores that produce distinctive lemon-shaped sporangia at their tip
which lead to further disease spread.

P. capsici is a soil borne, hemibiotroph pathogen that favors warm and wet
environments (Erwin and Ribeiro, 1996; Hausbeck and Lamour, 2004). The optimum
temperature for disease development is 25 to 28 °C. Multiple cycles of inoculation occur
in a single growing season. The asexual sporangia can germinate either directly or
indirectly (Katsura and Miyazaki, 1960). In direct germination, hypahe are produced
from the sporangia wall and penetrate through openings, including stomata, lenticels or
wounds. In indirect germination, also called zoosporogenesis, sporangia produce asexual
motile zoospores, which are the primary infection source during the growing season.
Temperature can inﬂuence whether sporangia undergo direct or indirect germination in P.
infestans (Judleson and Blanco, 2005).

Zoospores, can be spread by irrigation, rain, and free surface water on plants. The

wall-less zoospores swim by using two ﬂagella and are directed chemotactially,

electrotactically, or autoaggragationally to a primary infection site of the host plant
(Hardham, 2007; Latijnhouwers et al., 2003; Tyler, 2002). Chemoattractants can be non-
speciﬁc such as amino acids, or speciﬁc such as the isoﬂavones daizen and genistein
from soybean roots which only attract Phytophthora sojae. Electrotaxis is a response to
ion exchange carried at the surface of root that creates an electrical ﬁeld around the
rhizosphere (Morris and Gow, 1993). Autoaggragation in which zoospores congregate to
maximize infection rate, is thought to be species speciﬁc and could be related to calcium
released by the cyst (Reid et al., 1995; Tyler, 2002).

Soon after contact with the plant surface, zoospores shed their ﬂagella and encyst.
Encystment can be induced by many factors including chemical and physical stimuli,
temperature, calcium and phosphatic acid. However, the exact mechanisms which
governs zoospore movement, taxis, and encystment are not well known. Upon
encystment, zoospores secret adhesive material to help the cysts to adhere to the plant
surface. In the case of P. inﬁzstans, the adhesive material secreted by P. inﬁzstans has
homology to human mucins, suggesting a role in protection of the germiling from drying
or physical damage (dehardet et al., 2000).

Encystment is followed by germination of the germ tube and appressorium
formation which also directed by chemotaxic and thigmotropic growth. Periclinal or
anticlinal walls on epidermis surface and stomatal complex are usually targeted for
penetration (Hardham, 2007). Germ tubes often swell to form appressoria, which are
smaller and not melanized compared to fungal appressroia. Whether these appresoria

exert mechanical pressure on the plant surface is still unknown. Phytophthora capsici

can penetrate directly through the plant surface or through natural openings such as
stomata (Katsura and Miyazaki, 1960).

In case of Phytophthora palmivora, appressorium formation in vitro was highly
inﬂuenced by topographical signals, substrate hydrophobicity and nutrient conditions,
where rough, hydrophibic, and lower nutrient artiﬁcial surface promoted appressoria
formation (Bircher and Hohl, 1997). In potato leaf and P. infestans interaction, short
germ-tubes and appressoria were associated with rapid and successful penetration
whereas longer and aberrant germ-tubes were associated with unsuccessful infection or
incompatible plant-pathogen interaction (Grenville-Briggs et al., 2008).

Pathogenesis related factors including cell wall degrading enzyme such as
cutinases, polygalactumonases, pectate lyases, and cellulase are thought to be produced
by the germ tubes and appressoria (Judelson and Blanco, 2005; Latijnjousers et al., 2003).
These enzymes are thought to help penetration of plant surface. They also can be
recognized as elicitors which trigger defense response by both host and non-host plants
(Hardham, 2007; Judelson and Blanco, 2005). After successful penetration of the host,
hyphae grow intracellulary and develop haustoria, a specialized structure which obtain
nutrients from host (Latijnhouwers et al., 2003; O’Connell and Panstruga, 2006).

In addition, P. capsici produces a unique thick walled sexual spore called an
oospore, which enables it to overwinter. Two compatible mating types, A1 and A2, are
required for oospore formation (Erwin and Ribeiro, 1996). Both mating types produce
antheridium (male) and oogonium (female), and come into physical contact, antheridium
fertilize oogonium, subsequently develops oospores. In case of P. infestance, oospore

production is triggered in response to low temperature (Mosa et al., 1991 ).

Phytoplrthora capsici control in cucumber production

Controlling P. capsici in cucumber production has been difﬁcult. Oospores are
able to survive in the soil for more than ﬁve years, making crop rotation an inadequate
control method (Lamour and Hausbeck, 2001a, 2002, 2003; Hausbeck and Lamour,
2004). Also, P. capsici is heterothallic, therefore there is a chance of recombination to
introduce new variants that carry new virulent factors, contributing to the difﬁculty in
controlling this disease (Erwin and Ribeiro, 1996; Lamour and Hausbeck, 2003). Heavy
dependence on chemical control has resulted in the development of resistance to a
fungicide commonly used in Michigan, Ridomil® (active ingredient mefenoxam)
(Lamour and Hausbeck, 2000). In addition, since many farms do not practice rotation
with non-susceptible crops, the ﬁelds can build up high spore populations and develop
resistance to fungicides sooner.

Genetic resistance would be the optimal method of disease control. Cucumber
fruits are the most susceptible part of the plant (Hausbeck and Lamour, 2004). Cucumber
vines will look healthy at time of harvest, however the fruits can be heavily infested by P.
capsici (Hausbeck and Lamour, 2004). Therefore, Hausbeck et a1. (2002) initiated
screening for fruit resistance, but no signiﬁcant resistant genotype was identiﬁed out of
238 genotypes screened. Subsequent screening, including acessions tested as part of this
work (chapter 2) using a diverse collection of the cucumber gennplasm selected to
provide maximum genetic variance based on geographical distribution and isozyme data
(Knerr et al., 1989), has not identiﬁed any resistant genotypes to date.

It is therefore important to develop an integrated system for P. capsici control.

For example, incorporating cover crops to suppress the pathogen from spreading by

minimizing rain splash spore dispersal have been tested, however this strategy is difﬁcult
to reach an economically feasible level (Ristaino et al., 1997; Wang and Ngouajio, 2008).
Modifying canopy conditions to be less favorable for P. capsici development may also
facilitate disease control. Fruit are usually located under the canopy where the
environment is moist, warm, and close to the pathogen in the soil, thus favoring
conditions for disease development. Furthermore, applying fungicides to the fruits is
difficult since the dense canopy is covering the fruit. Studies with trellised cucumber
demonstrated reduced infection by Rhizoctonia solani, presumably due to higher air
circulation, better fungicide distribution, and less plant parts contacting soil (Hanna et al.,
1987; Konsler and Strider, 1973). However, trellising is expensive and labor intensive,
making it unusable for commercial pickling cucumber production (Russo et al., 1991).
Wider spacing to reduce disease pressure has been proposed, and this approach remains
to be tested (N gouajio et al., 2006). Another possible approach is to implement alternate
plant architectures for a less dense canopy to allow for increased air circulation and light

penetration or reduce fruit contact with the soil.

Genetic modiﬁcation of plant architecture for disease control

The idea of controlling disease by reducing canopy density with altered plant
architecture has been evaluated for other crops and diseases. One attempt was made by
Halterlein et al. (1981) for R. solani control on cucumber. They sprayed herbicide over
the canopy after the ﬁrst ﬂower to reduce the canopy density, and observed a signiﬁcant
reduction in the disease occurrence. Therefore, the authors concluded that the

microclimate under the reduced canopy created unfavorable conditions for the disease

development. A reduced canopy was presumed to allow for better spray distribution, less
humidity and increased light penetration, which together could contribute to disease
reduction (Halterlein et al., 1981). In addition, in earlier published work, Hamish (1965)
reported that continuous bright light inhibited oospore formation, while, continuous low
intensity light and complete darkness did not inhibit oospore formation. While spraying
herbicide on to a crop at the time of fruit development may not be a desirable production
strategy, other methods such as breeding for traits that confer to reduced canopy density
may be helpﬁrl. This was explored in chapter 2.

There are limited documented examples of plant breeding efforts which have used
an architectural approach to control disease incidence (Ando et al., 2007). Two of the
better documented examples are effect of canopy structure on white mold in bean and
effect of plant height on fusarium head blight of wheat

White mold (Sclerotinia sclerotiorum) is a common necrotrophic and soilbome
disease affecting dry bean (Phaseolus vulgaris) production worldwide. It favors cool and
moist environment and affects stems and pods especially during the stages of ﬂowering
and pod ﬁlling stages (Fuller et al., 1984). Steadman et al. (1973) compared disease
occurrence in nearly-isogenic lines differing in determinancy and in relation to plant
spacing. All genotypes tested showed signiﬁcantly lower disease occurrence in wider
spaced plots, and also determinate lines had lower disease occurrence than indeterminate
lines regardless of planting density. In addition, trellis-grown plants had higher yield and
lower white mold incidence, presumably due to modiﬁed microclimate which minimized
optimal conditions for white mold occurrence and increased photosynthesis by allowing

sun exposure (Coyne et al., 1974).

Height and leaf distribution inﬂuenced the white mold severity even within
upright growth habit beans, since taller and more open canopy upright genotypes had
lower disease incidence; whereas upright genotypes with dense canopy near the soil
surface had higher disease occurrence (Schwartz et al., 1987). These results suggest that
plant height and canopy density are also contributing to disease occurrence. By
combining protective spray application, disease control in upright growth habit genotypes
can be further improved (Schwartz et al., 1987).

The relationship between agronomic traits including growth habit, days to ﬂower,
canopy height and width, branching pattern, loading, days to maturity, seed size, and
yield and resistance to white mold was studied by Kolkman and Kelly (2002). Although
the agronomic traits associated with disease severity differed among the locations, the
most important trait was indeterminate growth habit. Based on the study, they proposed a
plant ideotype that has indeterminate bush growth habit, lodging resistance, and medium
canopy height, width, and branching pattern.

These results suggested the possibility of modiﬁed plant architecture as a means
to promote disease avoidance mechanisms for disease control. To facilitate white mold
control, bean breeders have selected for elevated canopy of an upright indeterminate bean,
and the open porous canopy from determinate bean (Coyne et al., 1974; Blad et al., 1978;
Fuller et al., 1984; Park, 1993). With the combination of alternating plant architecture
and better cultural practice, bean breeders were able to improve disease control and
increase yield.

In the case of fusarium head blight (FHB) of wheat (T riticum aestivum),

researchers observed that tall winter wheat lines resulted in lower FHB severity than

shorter lines, presumably since the location of the ear of tall lines is further from the soil
line where the inoculum is located (Mesterhazy, 1995). Hilton et al. (1999) also observed
that tiller height and severity of F HB was negatively related. Near-isogenic lines with the
dwarﬁng gene, RhtI , showed signiﬁcantly higher disease severity compared with the
lines without Rhtl, regardless of the genetic background. Comparing microclimate
differences between the tall and the short lines by measuring relative humidity at ear
height revealed no signiﬁcant difference in relative humidity, suggesting that the reduced
disease incidence may be more directly related to distance from the soil than modiﬁed
canopy conditions. F3 families of a cross between tall and short parents co-segregated for
disease resistance and tall straw, further indicating that genes controlling straw height
could be linked to FHB resistant quantitative trait loci.

Tan spot caused by Pyrenophora tritici-repentis, is a common disease in durum
wheat (T riticum turgidum) (De Wolf et al., 1998). The pathogen overwinters in old crop
residue. Ascospores, the primary inoculum, are released from the residues under rain,
high humidity, and a temperature above 10 °C. The secondary inoculum, conidia, is
dispersed by wind and germinates under prolonged leaf wetness (6 hr). Tan spot severity
was considered to be related to plant height as was observed for FHB. Fernandez et al.
(2002) tested this hypothesis by using near isogenic lines and randomly selected lines
differing in height. All the tested lines were susceptible to tan spot; however, the short
lines were more diseased than the tall lines. Furthermore, the plant height and leaf area
index were negatively correlated; the short lines had a denser canopy due to closer

position of lower leaves of adjacent plants. The denser canopies in the short lines might

also cause more favorable environmental conditions for infection, since there was no
correlation between height and disease severity under controlled inoculation.

In cucumber, a variety of growth habits exists within the cucumber germplasm
(Pierce and Wehner, 1990), however these traits have not been tested for the purpose of
controlling disease by architecture. There are 1,352 plant introductions (P18) in the
collection of the US. National Plant Gennplasm System (NPGS) which might be sources
of new plant architectures. In addition, other tools, such as nearly isogenic lines differing
in determinancy habit and leaf size have been developed (Serce et al., 1999). The growth
habit of these cucumbers might increase air circulation, light penetration, or accessibility

of the applied fungicide to fruit under the canopy.

Age-related resistance

Age-related resistance (ARR), also called adult plant resistance or
developmentally related resistance, is becoming increasingly recognized as an important
component of plant defense against infection (Develey-Riviére and Galiana, 2007; Panter
and Jones, 2002; Whallen, 2005). Increased resistance has been observed as young
tissues develop in many plant-pathogen systems, including diseases caused by fungi,
oomycetes, bacteria, nematodes, and viruses. ARR has been observed for P. capsici
infection of pepper (Capsicum annum) plants (Kim et al., 1989). The reduced
susceptibility was suggested to be related to physiological changes in root and stem
tissues.

Once ARR is induced, the resistance persists for the rest of the plant life until

senescence. ARR is distinct from the increase in susceptibility which occurs in

10

association with ripening or senescence in old, mature organs. ARR can be non-speciﬁc,
increasing overall disease resistance to multiple pathogens, it can increase resistance to
all strains of a particular species, or can be speciﬁc to a given pathogen strain. Little is
known about the mechanisms responsible for ARR.

There is evidence that salicylic acid is important to ARR in the Arabidopsis
response to Pseudomonas syringae (Kus et al., 2002) and Hyaloperonospora parasitica
(McDowel et al., 2005). ARR in rice (Oryza sativas) to Xanthomonas spp. may be
related to developmentally-regulated R gene-mediated resistance which is controlled
post-transcriptionally, or by other mechanisms (Century et al., 1999; Koch and Mew,
1991; Mazzola et al., 1994). Developmentally-regulated expression of single-gene
mediated resistance also has been observed for potyvirus infection of cucumber seedlings
(U 11311 and Grumet, 2002; Wai and Grumet, 1995).

AR can be induced at major transitions occurring during a plant’s life. For
example, ARR has been observed at developmental transitions including juvenile to adult,
or vegetative to ﬂowering (O’Connell and Panstruga, 2006). Disease resistant or
susceptible mutants have been identiﬁed that have altered developmental processes;
conversely, mutants in developmental functions can have altered defense capability,
suggesting the possibility that resistance gene products maybe evolutionary related to
developmental gene products, or that developmental genes may confer resistance or
induce processes that confer resistance (Whalen 2005). For example, the ARR to
common rust caused by Puccinia sorghi is delayed in Congrass] (CgI) maize (Zea mays)
mutant which has an extended juvenile-vegetative phase. Cg] mid-whorl leaves with

juvenile traits were highly susceptible, while the wild type mid-whorl leaves were

11

resistant (Abedon and Tracy, 1996). Also, the vegetative phase of tobacco (Nicotiana
tabacum) was highly susceptible to Phytophthora parasitica, however plants become
resistant at the time of the ﬂowering phase transition (Hugot et al., 1999). Non-salicylic
acid (SA) expressing transgenic (N ahG) tobacco plants showed that P. parasitica
infection of the leaves does not require SA accumulation, however, infection after ﬂoral
transition requires SA accumulation. In addition, Arabidopsis showed ARR to
cauliﬂower mosaic at the transition from vegetative phase to ﬂoral phase due to
developmental changes affecting on the long-distance virus movement (Leisner et al.,
1993).

Some plants only develop ARR in speciﬁc tissues or organs. Scab caused by
Venturia inaequalis on apple (Malus domestica) leaves showed ARR, young apple leaves
are more susceptible than old leaves (Li and Xu, 2002). Also, in the case of soybean
(Glycine max), hypocotyl age inﬂuenced the susceptibility to P. sojae (Lazarovits et al.,
1980)

AR also has been observed in fruits. Developing grape berries (Vitis vinifera)
showed decreased susceptibility to infection by Unicinula necator, causal agent of
powdery mildew, and to Plasmopara viticola, causal agent of downy mildew (Ficke et al.,
2002; Gadoury et al., 2003; Kennelly et al., 2005). Berries are highly susceptible for the
ﬁrst several weeks after anthesis. Developmental changes in berries, such as brix content
or morphological change of stomata to lenticels were thought to be related to
susceptibility changes, however, the mechanism has not been determined. In the process
of screening for P. capsici resistance in cucumber I also observed age-related resistance

in the developing fruit. This observation was studied in chapter 3.

l2

Genomic approaches to study fruit development in cucumber

Analysis of gene expression during cucumber fruit development may provide
further insight into the cucumber—P. capsici interaction. Cucmnber genomic tools,
however, have been very limited. The number of cucumber ESTs in the National Center
for Biotechnology Information (NCBI) database is only approximately 8,000 (4/15/2009
searched) compared to other species such as Arabidopsis - 1,728,728, rice - 1,260,123,
and tomato - 261,630. In the past year, however, the cucumber genome was reported to
be sequenced by a group at the Chinese Academy of Agriculture Science and the Beijing
Genomics Institute, Shenzhen (Huang et al., 2009). It is anticipated that the 365 Mb
sequenced genome will be publicly available in the near future. Having a sequenced
genome and generating more ESTs will be a tremendous advantage, not only for contig
assemblies and gene annotations, but also for providing tools to study complicated
biology of plants.

The development of next generation sequencing technologies has dramatically
improved sequencing capability with respect to amount of data, time, and cost (Mardis,
2008; Margulies et al., 2005). Roche (Branford, CT) introduced 454 pyrosequencing
technology (Margulies et al., 2005; Droege and Hill, 2008) which can sequence over 100
Mb for 7.5 hrs with average reading of 200 bp. A new version of 454, GS FLX Titanium
Series reagent, was introduced in 2008 which can sequence over 400 Mb with average
reading of 400 bp. The length of reads generated by 454 pyrosequencing allow for contig
assemblies and gene annotations of poorly characterized genomes. In addition, one of the
applications of the new sequencing technology is gene expression analysis, since the

number of contig reads represents frequency of the particular gene expression (Eveland et

13

al., 2008; Ohtsu et al., 2007; Torres et al., 2008). 454 sequencing also has capability of
loading multiple samples for a run, allowing side-by-side analysis. Gene expression
analyses by 454 pyroseuquencing results have been correlated with microarrays, northern
blot analysis or quantitative real time polymerase chain reaction (qRT-PCR) assays,
suggesting high reproducibility and accountability for gene expression analysis (Eveland
et al., 2008). It also can be used as an alternative to microarrays, especially for crops
with small ESTs numbers and/or no microarray platform available, such as cucumber.

Model plants, such as Arabidopsis thaliana and rice (0. sativas) are well studied
in many aspects of plant development with the aid of tremendous information generated
by genomic tools. Fully sequenced genomes, microarrays, and large numbers of ESTs
and mutants are available. However, these species are not well suited to address
questions related to ﬂeshy fruit development. In case of ﬂeshy fruits, tomato (Solanum
lycopersicum), apple, and grape (V. vinifera) are well studied (Carrari and Femie, 2006;
Giovannoni, 2004; J anssen et al., 2008; Lemaire-Chamley et al., 2005; Moore et al.,
2002; White, 2002). The primary emphasis of mu studies has been on late stage of
development and important fruit quality attributes associated with maturation and .
ripening (Lee et al., 2007; Lemaire-Chamley et al., 2005).

There has been less study done on early ﬂeshy fruit development, though this
stage is essential for all fruit. For example, in tomato fruit, many new genes, and many
genes with no known function were recovered from young tomato fruit cDNA libraries
from tissues at ranging in 8, 12, and 15 days post anthesis (DPA). In addition, reﬂective
of less study of these stages, 8 DPA fruit had the highest proportion of genes with no

known function (Lemaire-Chamley et al., 2005). There are three main phases of early

14

ﬁ'uit development; fruit set, cell division, and cell enlargement (Gillapsy et al., 1993). As
might be expected, genes that were highly expressed in young developing tomato fruit
locules and mesocarp included those with predicted functions in Mt growth, fruit
protection, photosynthesis, and cell expansion (Lemaire-Chamley et al., 2005). In the
case of apple, genes with functions associated with control of cellular organization, cell
cycles, photosynthesis, protein synthesis had higher expression in early fruit (Janssen et
al., 2008; Lee et al., 2007). Many genes had similar patterns of expression in tomato and
apple, suggesting that with more increased EST data from other species it will allow
better comparison and identifying common fundamental processes in fruit development
(Janssen et al., 2008). Cucumber fruit development and gene expression was explored in

chapter 4.

Objectives of dissertation

There are numerous difﬁculties limiting establishment of effective methods to
control P. capsici in cucumber production as described above. In this dissertation, my
ﬁrst objective was to examine alternative cucumber architecture as a means to control P.
capsici occurrence in cucumber production. Nearly-isogenic lines differing for
determinate grth habit and leaf size were tested for disease occurrence. In addition, a
sample of the cucumber germplasm, including the cucumber core collection and
additional PIs that have been annotated in the NPGS database for possible short
intemodes or bush growing habit, were screened for additional architectural variation.

Promising variants were then tested for disease occurrence in infested ﬁeld plots.

15

I also performed additional screening for genetic sources of resistance to P.
capsici. During screening for resistance, fruit age/size appeared to be related to
susceptibility. Therefore, I examined the effect of fruit age on susceptibility to P. capsici
in greenhouse- and ﬁeld-grown cucumber and other cucurbit crops including melon
(Cucumis melo), watermelon (Citrullus lanatus), butternut squash (Cucurbita mocshata),
and four Cucurbita pepo crops, zucchini, yellow summer squash, acorn squash, and
pumpkin. I also examined possible roles of the cucumber fruit surface properties in age
related resistance.

Lastly, to understand the basis of the age-related resistance in cucumber fruit-P.
capsici interaction, and to increase understanding of early fruit development in cucmnber,
I performed morphological and global gene expression analyses. Hand-pollinated
greenhouse grown cucumber fruit ranging 0-32 DPP were used to catalogue
morphological changes associated with cucumber fruit development and to develop
cDNA libraries for 454 pyrosequencing. Rapidly expanding 8 DPP fruit were

characterized for gene expression.

16

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23

Chapter 2

Ando, K. and R. Grumet. 2006. Evaluation of altered cucumber plant architecture as a

means to reduce Phytophthora capsici disease incidence on cucumber fruit. J. Amer. Soc.
Hort. Sci. 131:491-498.

24

Evaluation of altered cucumber plant architecture as an approach to reduce
Phytophthora fruit rot
Abstract
Fruit rot induced by Phytophthora capsici Leonian is an increasingly serious

disease affecting pickling cucumber (Cucumis sativus L.) production in many parts of the
United States. The absence of genetically resistant cultivars and rapid development of
fungicide resistance makes it imperative to develop integrated disease management
strategies. Cucumber fruit which come in direct contact with the soil-home pathogen are
usually located under the canopy where moist and warm conditions favor disease
development. We sought to examine whether variations in plant architecture traits that
inﬂuence canopy structure or fruit contact with the soil could make conditions less
favorable for disease development. As an extreme test for whether an altered canopy
could facilitate P capsici control, we tested the effect of increased row spacing and trellis
culture on disease occurrence in the pickling cucumber 'Vlaspik'. Temperature under the
canopy was lowest in trellis plots, intermediate in increased spacing plots, and highest in
control plots. Disease occurrence in the trellis plots was signiﬁcantly lower than in other
treatments, indicating that preventing fruit contact with the soil reduced disease
occm'rence. The effect of currently available variation in plant architecture was tested
using nearly-isogenic genotypes varying for indeterrrrinate (De), determinate (de),
standard leaf (LL), and little leaf (ll) traits. Plants with standard architecture had higher
peak mid-day temperatures under the canopy and greater levels of P. capsici infection;
however, levels of disease occurrence were high for all genotypes. Screening a collection

of approximately to 150 diverse cucumber accessions identiﬁed to serve as a

25

representative sample of the germplasm, revealed variation for an array of architectural
traits including main stem length, intemode length, leaf length and width, and number of
branches; values for 'Vlaspik' were in the middle of the distribution. Plant architectures
that may allow for more open canopies, including reduced branching habit and compact
growth, were tested for disease incidence. One of the compact lines (PI 308916), which
had a tendency to hold young fruit off the ground, exhibited lower disease occurrence.
The reduced disease occurrence was not due to genetic resistance, suggesting that
architecture which allows less contact of fruit with the soil could be useful for P capsici

control for pickling cucumber.

Introduction

Fruit rot caused by the Oomycete pathogen, Phytophthora capsici is currently one
of the most serious diseases affecting cucumber (Cucumis sativus L.) production in many
parts of the United States (Hausbeck and Lamour, 2004). In Michigan, the top pickling
cucumber producing state, pickling cucumber acreage has increased over the past ﬁve
years (Michigan Department of Agriculture, 2001), however, maintaining and increasing
yield has become difﬁcult due to losses caused by P. capsici.

P. capsici was ﬁrst reported by Leonian (1922) as a host speciﬁc pathogen of chili
pepper (Capsicum annum L.) occurring in New Mexico. Later, it was found to infect a
wide variety of hosts ranging from woody plants and vegetable hosts (Erwin and Ribeiro,
1996). Although incidence of cucumber infection was ﬁrst reported in Colorado in 1936

(Kreutzer, 1937), it has only recently become a common problem in cucumber production

26

areas (Babadoost, 2004; Hausbeck and Lamour, 2004). Once cucumber ﬁelds are
infected by P. capsici, severe yield loss due to infection of the fruit is frequently observed
(Babadoost, 2004; Hausbeck and Lamour, 2004). Cucumber fruit infected by P. capsici
typically develop a depressed ﬁ'uit surface with a water soaked appearance followed by
white powdery mycelium covering the affected region.

Optimal growth conditions for P. capsici include a warm and wet environment,
particularly around 25 to 30 °C (Hausbeck and Lamour, 2004). P. capsici sporangia have
a recognizable lemon shape and produce asexual motile zoospores which are the primary
cause of infection during the growing season, since they can be spread by irrigation, rain,
and free surface water on plants (Erwin and Ribeiro, 1996; Hausbeck and Lamour, 2004).
In addition, P. capsici produces a unique thick walled structure called an oospore, which
enables it to overwinter. Since oospores are able to survive in the soil for more than ﬁve
years, crop rotation becomes impractical, contributing to the difficulty in controlling this
disease (Lamour and Hausbeck, 2001a, 2002, 2003).

Heavy dependence on chemical control has resulted in the development of
resistance, as has been identiﬁed with respect to mefenoxam (Ridomil Gold; Novartis,
Greensboro, NC), a fungicide commonly used in Michigan (Lamour and Hausbeck, 2000,
2001a, 2001b). In addition, since many farms do not practice rotation with non-
susceptible crops, spore populations can increase, and sexual recombination among
mating types can allow for transfer of resistance genes (Lamour and Hausbeck, 2000,
2001a, 2001b, 2002). Gevens et al. (2006) screened 482 C. sativus accessions for fruit
resistance, but no signiﬁcant source of resistance has been identiﬁed to date.

Consequently these conditions make it necessary to seek alternative control strategies.

27

Field observations show that cucumber fruits are susceptible to P. capsici, while
roots or crowns are much less susceptible. Fields will frequently appear healthy at the
time of harvest as judged by vegetative growth, but the fruits, which are usually located
under the canopy, can be heavily diseased (Hausbeck and Lamour, 2004). The
environment under the canopy is moist and warm, and the fruits are in close proximity to
the soil-bome pathogen, thus favoring conditions for disease development. Also, the
dense canopy covering the fruit prevents fungicides from reaching them, making control
more difficult.

These observations suggested that it might be possible to reduce the problem by
reducing canopy density (i.e., temperature, or humidity) by altering cultural conditions
such as spacing or trellising, or with altered plant architecture. Cucumber disease control
by trellis culture has been studied for Rhizoctonia solam’ Kuhn (Konsler and Strider, 1973;
Hanna et al., 1987). Studies showed that trellised cucumber had less R. solani occurrence
presumably due to higher air circulation, better fungicide distribution, and reduced
contact with the soil (Hanna et al., 1987; Konsler and Strider, 1973). However, trellising
is expensive and labor intensive, making it unsuitable for commercial pickling cucumber
production (Russo et al., 1991).

The effect of reducing cucumber canopy density on infection by R. solani also
was tested by herbicide treatment applied after initial ﬂowering (Halterlein et al., 1981).
The herbicide treated plots exhibited a signiﬁcant reduction in disease occurrence,
leading the authors to conclude that the microclimate under the reduced canopy
contributed to disease reduction by allowing for better spray distribution, less humidity

and increased light penetration.

28

Altered canopy structure may also be achieved by modiﬁed plant architecture.
This strategy for disease control has been utilized in bean (Phaseolus vulgaris L.)
breeding (Coyne, 1980). White mold [Sclerotinia sclerotiorum (Lib.) de Bary] is a
common disease affecting dry bean production. To facilitate white mold control, bean
breeders have selected for elevated canopy of a prostrate indeterminate bean, upright
canopy of indeterminate bean, and open, porous canopy from determinate bean (Blad et
a1, 1978; Coyne et al., 1974; Fuller et al., 1984; Kolkman and Kelly, 2002; Park, 1993).
With the combination of altered plant architecture and improved cultural practices, bean
breeders were able to improve disease control and increase yield.

F ortrmately, a variety of growth habits exists within the cucumber gennplasm
(Pierce and Wehner, 1990). Some genotypes produce shorter vines, others have smaller
leaves, or various fruit set positions. Determinate plants possessing the de gene, have
shorter vines, shorter intemode length, and less lateral branches (George, 1970; Grumet
and Duvall, 1993; Wehner, 1989). The little-leaf trait, incorporated into breeding line H-
19, is controlled by a single-gene mutantion (II) and is characterized by small leaves, a
highly branched growth habit with shorter vines and smaller stems (Goode et al., 1980;
Schultheis et al., 1998). The compact trait, an extreme dwarf plant type, is controlled by
the homozygous recessive gene cp, which has short intemodes, poorly developed tendrils,
and small ﬂowers (Kauffrnan and Lower, 1976).

F our nearly isogenic pickling cucumber lines were developed by Serce et al.
(1999) that differ for vine development (indeterminate and determinate), branching habit,
and leaf size (standard and little-leaf). These nearly isogenic lines, which minimize other

genetic inﬂuences, can be used to test effects of plant architecture. Serce et al. (1999)

29

tested the response of these lines to water stress; however, they have not been tested for
effect on disease development. The growth habit of these cucumbers might increase air
circulation, light penetration, or accessibility of the applied fungicide to fruit under the
canopy.

In addition to testing existing architectural variants, identiﬁcation of new plant
architecture also could be valuable for inﬂuencing disease development. There are 1,3 52
plant introductions (P13) in the collection of National Genebank, the US. National Plant
Gennplasm System (NPGS). Knerr et al. (1989) identiﬁed a group of 100 P13 which
serve as a representative sample of the germplasm with maximum genetic variance based
on geographical distribution and isozyme data.

In this study, we examined the possibility of an architectural approach to
minimize P. capsici occurrence in pickling cucumber production. Three types of studies
were performed. The ﬁrst study was the evaluation of the effectiveness of trellis culture
on controlling P. capsici, as an extreme test of the hypothesis that altered plant
architecture may facilitate control of P. capsici, since the intended method of disease
control by altered canopy structure (increased air movement, reduced humidity, or
reduced fruit contact with the soil) is similar to that for trellis culture. The second study
utilized the nearly isogenic lines differing for architectural traits of determinate grth
habit and leaf size developed by Serce et al. (1999). The third study screened a sample of
the cucumber gennplasm for additional architectural variants using the collection
classiﬁed by Knerr et al. (1989), and an additional 50 HS that have been annotated in the

NPGS database for possible short intemodes or bush growing habit.

30

Materials and Methods
Trellis study

The trellis study was performed on a P. capsici-infested ﬁeld at the Michigan
State University (MSU) Muck Farm (Bath, MI) during the summer of 2003 (planting date,
May 30, 2003). The commercial pickling cucumber cultivar ‘Vlaspik’ (Seminis
Vegetable Seed Inc., Oxnard, CA) was used for all treatments and borders. Three
treatments, standard spacing (0.5 m between rows), wide spacing (1.5m between rows),
and trellis (1.5m between rows), were arranged in a randomized complete block design
with four replications. All plots were 4.6 m wide and in-row spacing was 0.1 m. The
standard spacing plots consisted of ten, 3 m—long rows, the wide spaced and trellis plots
had three, 3 m-long rows. Fruits were taken from center 2.4 m x 1.2 m of the standard
spacing plot and wide spacing plots, and center 2.4 m of the middle row of the trellis plot.

The trellis plots were constructed with 1.5 m high stakes placed at 1.2 m intervals
within the rows. Plastic mesh was attached to braided nylon ropes which were placed
parallel to the row at the top and bottom of the stakes. Cucumbers were trained to the
plastic mesh with support of twine or their own tendrils. Irrigation was provided by rain
or overhead sprinkler to 25 mm a week. Prior to harvest, irrigation was increased to 75-
100 mm a week to stimulate infection.

Temperature under the canopy was taken every 15 minutes using WatchDog data
loggers (Spectrum Technologies Inc., Plainﬁeld, IL) from the time of ﬁrst female ﬂower
bloom until second harvest for three replications. The data loggers were placed within
the canopy at positions where fruits were located. The temperature data was analyzed by

analysis of variance (AN OVA) using SAS proc mixed procedures (SAS Institute Inc.,

31

 

3?;

lillC

Arc

 

Cary, NC).

Fruits were harvested four times. Because disease frequently becomes more
apparent during storage, the fruit was inspected for disease at the time of harvest and then
re-evaluated after four days of storage in plastic crates in the barn. Yield and disease
occurrence were analyzed by AN OVA using SAS proc mixed procedures. Samples of
infected fruit were collected from each harvest to verify presence of P. capsici according
to the method of Lamour and Hausbeck (2003). Brieﬂy, infected fruits were sanitized
with 70 % ethanol and dissected to remove 0.25 cm3 pieces from the margin of the
infected area. The epidermis was removed, pericarp pieces were placed on BARP
medium (Lamour and Hausbeck, 2003), and incubated at room temperature under the
light for 3 — 4 days until sporulation was observed. Fungal samples were observed by
microscope for the presence of distinctive lemon shaped sporangia.

Architectural study with nearly-isogenic lines.

The architectural study was planted at the ﬁeld described above on June 27, 2003,
using a set of four nearly-isogenic pickling cucumber lines developed by Serce et al.
(1999) to vary for architectural features: 1) indeterminate and standard leaf (DeDeLL), 2)
determinate and standard leaf (dedeLL), 3) indeterminate and little leaf (DeDeII), and 4)
determinate and little leaf (dedell). Five additional lines were included: H19 (II)
(standard little leaf type - H19 was used as the parent to introduce the little leaf trait into
the nearly isogenic lines described above); compact (cpcp) (J. E. Staub, personal
communication); Marketmore 76 and Marketmore 86 (nearly-isogenic indeterminate (76)
and determinate (86) cultivars); and Vlaspik (standard commercial cultivar, indeterminate

and standard leaf).

32

The genotypes were planted in 1.8 m x 3.0 m plots with 0.5 m row spacing with
four plants per 30 cm to approximate local commercial conditions. Each plot was paired
with the standard commercial cultivar, ‘Vlaspik’, in order to minimize effect of potential
variation in disease pressure throughout the ﬁeld. The pairs of plots were planted in a
randomized complete block design with four replications. Canopy data collection,
harvest, disease analysis, and P. capsici veriﬁcation were as described above.

Screening for architectural variants

The architectural screening experiment was performed in a non-replicated trial on
non-P. capsici infested soil at the MSU Horticulture Research and Teaching Center (East
Lansing, MI) during the summer of 2003 (planting date, May 20, 2003). Cucumber
accessions were supplied by the North Central Regional Plant Introduction Station, Ames,
Iowa. The Pls were sown in the greenhouse and transplanted to the ﬁeld at 14 days.
Irrigation was provided by rain or overhead sprinkler to 2.5 cm a week.

To allow for observation of architecture, plants were spaced 0.5 m apart in 3.7 m
single-row long plots with 1.5 m between rows. One ‘Vlaspik’ plant was put at the
beginning and end of each plot to separate plots. Each row included one plot of ‘Vlaspik’
to allow for comparison of plant architecture. All plots were visually observed weekly
and plant architecture data was collected at approximately 60 days after planting for the
P18 which showed obvious visual differences from ‘Vlaspik’. Measurements were taken
from the middle three plants from each plot. Traits evaluated were: main stem and
intemode length, number and position of branches, leaf size, grth habit, and fruit
position relative to the soil. Main stem length was measured from the soil line to the

apical bud. Intemode length was measured for node positions 5 to 6 and 8 to 9 from the

33

base of the plant. Number of branches were counted for the main the main stem. Leaf
width and length were measured for fully mature leaves at node positions 5 and 8.
Correlations between traits were analyzed by SAS proc reg procedures.

Architecture study with selected P18 and reduced vine commercial lines

Several lines were selected for further evaluation based on architectural variation;
these included: PI 192940 (China), 227207 (Japan), 308915 (Russia), 308916 (Russia),
358814 (Malaysia), 401734 (Puerto Rico), and 466921 (Russia). Seeds for these
genotypes were multiplied in the greenhouse during spring of 2004. The above listed Pls
and four additional commercial cultivars described as having short vines, ‘Arabian,’
‘Colt,’ ‘Palomino,’ and ‘Stallion,’ were tested for their architectural effect on P. capsici
occurrence in the summer of 2004 on infested soil at the Muck Farm as described above
(planting date, June 21, 2004). ‘Vlaspik’ was used as control. Experimental design and
data collection were performed as described above for the nearly-isogenic lines trial,
except individual rather than paired plots were used, as the 2003 trial did not indicate
obvious non-uniformity in disease presence in the ﬁeld (data not shown). Architectural
data were collected as described above.

The fruits from tested P18 and ‘Vlaspik’ (control) were evaluated for P. capsici
resistance. Fruits were washed, air-dried, then inoculated with 10 day old mycelium of P.
capsici isolate OP97 (Gevens et al., 2006) and incubated at room temperature. Initial
fruit examination was made at 4 days post inoculation (DPI). Fruits were examined for
presence of water soaking symptoms (visible dark discoloration on fruit surface) or

sporulation (powdery mycelium on fruit surface).

34

Results
Trellis study

Canopy development varied among treatments in the trellis experiment. The
narrow-spaced plots achieved ﬁrll canopy closure one week prior to the ﬁrst harvest
(approximately 8 weeks post-planting). The wide-spaced plots continued to expand
throughout the season, but did not fully close even at the end of the experiment.
However, at the positions where the fruits were located, the canopy density above the
fruit appeared to be similar for the narrow-paced and the wide-spaced plots.

The temperature under the canopy at positions where fruit were located was
recorded at 15 minute intervals for approximately three weeks spanning the ﬁrst and
second harvest dates. The daily maximum temperature at fruit positions under the
canopy was similar for the narrow-spaced and wide-spaced plots, while the trellis plots
had a signiﬁcantly lower temperature (Table 2-1). Minimum temperatures among the
treatments followed the same trend. There was a progressive decrease in the average heat
units above 25 °C moving from the narrow-spaced plot, to the wide-spaced plot, and the
trellis plots.

As might be expected due to the greater number of rows, yields at the ﬁrst two
harvest dates were highest in the narrow-spaced plots (Figure 2-1A). Later in the season
(harvests 3 and 4) as the vines increased in size, yield of the wide-spaced and trellis plots
increased to match the narrow-spaced plots. Almost no fruit had obvious symptoms
(sporulation) of P. capsici at the time of harvest on the ﬁrst and second harvest dates;
however, at the third and fourth harvest dates, a small portion of fruits from the narrow

and wide plots showed infection at the time of harvest (Figure 2-1 B). The numbers of

35

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6 4 - -v- Trellis

 

 

 

30‘

20-

 

10-

 

 

Diseased fruit at 4 DPH (%) Disease fruit at harvest (°/o)
b
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Harvest

Figure 2-1. Mean cucumber fruit yield per aplot (A) and percent disease occurrence at
harvest (B) and 4 days post harvest (DPH) (C) at four harvest dates for standard, narrow,
and trellis plots. Each point is the mean of four replicate plots i SE.

37

infected ﬁ'uit increased when observed 4 days post harvest (DPH) (Figure 2-1C). Since
obviously infected fruits (i.e. sporulated) were removed prior to storage, the increased
percentages should reﬂect disease development of fruits that had been initially infected in
the ﬁeld. In the narrow plots, disease occurrence at 4 DPH increased as the season
progressed (Figure 2-1 B, C). Also, disease occurrence at 4 DPH in the wide plots
increased in synchrony with expansion of the canopy (Figure 2-1C). Although total yield
summed over the four harvest dates was signiﬁcantly higher for the narrow than the wide
plots, percent disease occurrence averaged over the four harvests was not signiﬁcantly
different (Table 2-1).

Disease occurrence in the trellis plots remained extremely low throughout the
season, and total disease occurrence at 4 DPH in the trellis plots was only 1.4 % (Figure
2-1C, Table 2-1). Even in the third and fourth harvests, when all plots were producing
similar amount of fruit (Figure 2-1A), disease incidence in the trellis plots was
signiﬁcantly lower (the percent infection averaged over the 3rd and 4th harvests at 4DPH
was 15.2 %, 16.5 %, and 1.9 % respectively for narrow, wide spacing, and trellis plots)
(Figure 2-1B,C).

Architectural study with nearly isogenic lines

There were visible differences in canopy structure and the time of canopy ﬁlling
among the genotypes tested in the architectural study with near-isogenic lines differing
for leaf size and determinacy. Despite smaller leaves, the II genotypes (H19, DeDelI, and
dedell) appeared to have very dense canopy, whereas de genotypes generally developed a
more open canopy based on visual observation. All fruits from the tested genotypes were

located on the ground. The genotypes with standard commercial architecture

38

(indeterminate growth habit and standard leaf size, DeDeLL, ‘Marketmore 76', and
‘Vlaspik’), tended to maintain higher temperature under the canopy than those with
nonstandard architecture (dedeLL, DeDelI, dedell, ‘Marketmore 86', H19 and compact).
Mean peak mid-day temperatures were signiﬁcantly higher for the standard types (30.9
°C) than the de, II, and cp (26.5 °C) types (LSD = 1.4, P S 0.05), suggesting that there was
an effect of the modiﬁed architecture on canopy conditions.

‘Vlaspik’ had signiﬁcantly higher yield at the ﬁrst harvest; however, the other
architectural types became comparable to ‘Vlaspik’ at later harvests (Figure 2-2A). One
of the lines, DeDeLL did not produce fruits and so was excluded from fruit analysis.
Disease distribution in the experimental ﬁeld was uniform based on disease occurrence in
‘Vlaspik’ plots (data not shown). Overall disease occurrence in this experiment was
higher than in the trellis expreriment. This is likely due to timing of the experiments; the
architecture experiment was carried out later in the season, where the higher temperatures
and increased secondary inoculum population led to more optimal conditions for P.
capsici infection. Disease occurrence at harvest increased throughout the season from 0-
10 % at the ﬁrst and second harvests to 15-45 % to the third harvest (Figure 2-ZB).

On average ‘Vlaspik’ showed higher disease occurrence than the other
architectural types at 4 DPH; however, disease occurrence at 4 DPH in all types was well
above economically-acceptable levels (approximately 30 %) (Table 2-2, Figure 2-2C).
Both ‘Marketmore 76' (DeDe) and 86 (dede) had higher disease occurrence at both 0 and
4 DPH compared to other genotypes over three harvest dates, suggesting that these
genotypes are more susceptible to P. capsici regardless of architectural effect (data not

shown).

39

 

50‘

401

30‘

20-

Fruit (nonlot)

10-

 

 

 

50 . —o— Vlaspik

....... OH”... Compact
40 _ _ — + — — Determinate
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10-

 

 

 

 

100 .

80-

 

60-

40-

20*

 

 

Diseased fruit at 4 DPH (%) Diseased fruit at O DPH (%)
q T
i

 

 

Harvest

Figure 2-2. Mean fruit number per plot (A), percent disease occurrence at harvest (B)
and four days post harvest (C) for ‘Vlaspik’, determinate (dedeLL and dedell), little leaf
(DeDell, dedell, and H-1 9), and compact architectural types. Each point is the mean of 4-
28 plots (4 replicate plots per genotype) 1 SE

40

Table 2-2. Percent Phytophthora capsici-infected cucumber fruit rated at 4 days
pgtharvest and averaged over three harvest dates.

 

 

Architecture type Total disease occurrence (%)
Standard (‘Vlaspik’) 68.2bz

Little leaf (DeDell, dedell, and H-19) 38.8a

Compact 34.1a
Determinate (dedeLL, and dedell) 28.9a

LSDons 17-2

 

zValues followed by same letter are not Signiﬁcantly different at P S 0.05 (LSD).

41

Screening for architectural variants

Among the 150 accessions examined in the ﬁeld for architectural variation, ﬁfty
appeared to have distinctive architecture relative to ‘Vlaspik’ based on visual inspection.
These 50 P13 were measured for main stem length, intemode length, number of branches,
and leaf width and length. The PIs exhibited a normal distribution for the measured traits
which varied in magnitude by 2- to lO-fold (Figure 2-3). Main stem length ranged from
20 to 250 cm, average intemode length from <1 to 9 cm, number of branches from <1 to
9, and leaf width from 8 to 19 cm. ‘Vlaspik’ was usually in the middle of the distribution.
There were strong correlations between main stem length and intemode length (P < 0.001)
and between leaf width and leaf length (P < 0.001) as might be expected, but not between
other traits (Table 2-3). An additional 110 accessions were observed in 2004, but no new
architecture traits were observed relative to the 2003 sample.

Several Pls were identiﬁed for further study at commercial planting densities. Pls
192940, 227207, 249562, 358814, and 401734 which had less branching and large
intemode and main stem length relative to ‘Vlaspik’, were categorized as reduced
branching type (R) (Table 2-4). The reduced branching types were selected due to their
open habit suggesting they may form a more open canopy, with less canopy coverage
over the fruits. PI 466921 had determinate growth habit with a short main stem (D) and
no branching; therefore less canopy coverage of the fruit was expected. PI 308915 and
308916 had short intemodes and upright growth habit (CP), and were similar to the
compact type tested above, but with a more extreme phenotype. Compact types also had
a tendency to hold young fruit off the ground, at least at the early stage of fruit

development, which might help reduce disease occurrence; this trait was more

42

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Table 2-3. Correlation (R2 value) between stern length, intemode length, leaf length and
width, and number of branches.

 

 

Main stem Intemode Leaf Leaf Number of
length length length width branches
Main stem length — 0576*" 0.032 0.101 0.002
Intemode — — 0.002 0.046 0.075
Leaf length — — — 0.708'“ 0.053
Leaf width — — — — 0.138

Number of branches — — _ _ _
***Signjﬁcant correlation at P < 0.001.

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45

pronounced for PI 308916.
Architecture study with selected P13 and commercial lines

The architecture types selected from the PI screening were tested in a P. capsici
infested ﬁeld at commercial planting densities. Four commercially available cultivars
described as having small plant size (Arabian, Colt, Palomino, and Stallion) also were
tested. However, in these conditions, vine length appeared to be comparable to ‘Vlaspik’.
We observed differences in main stem length and branching number for the selected PIs
compared to the initial screen in 2003 (Table 2-4). The PIs with reduced branching habit
and ‘Vlaspik’ generally had longer main stem length and more branching in 2004. The
compact and determinate lines had equivalent stem lengths in both years, with very short
vine lengths for the compact PI’s. In 2004, the reduced branching lines produced more
branches than had been observed in 2003 when they were more widely spaced.
Similarly, the compact lines showed a marked increase in branch number in 2004.

Several of the genotypes [compact (PI 308915 and P1308916), determinate with
no-branching, (P1466921), and reduced branching (PI 224207 and PI 358814) types] did
not germinate as uniformly as commercial genotypes and so these were excluded from
canopy analysis. However, there were visible differences in canopy structure among the
genotypes which had better germination rate. While the time of canopy ﬁlling and the
density of canopy among ‘Vlaspik’ and the four commercial small vine types appeared to
be comparable, the two reduced-branching types (PI 192940 and PI 4017 34), had a more
open canopy compared to ‘Vlaspik’ and the commercial lines as assessed by amount of

visible bare ground.

46

Earlier in the season, daily maximum temperatures under the canopy were higher
in reduced-branching genotypes than commercial architecture types. The mean maximum
temperatures from 27 July to 3 Aug. were 28.8 °C for reduced branching lines vs. 23.6 °C
for ‘Vlaspik’ (LSD=1.7, P S 0.05), suggesting that the more open canopy alloweddirect
sunlight penetration. As canopy ﬁlling progressed, this phenomenon diminished with
mean maximum temoeratures of 21.9 °C for the reduced branching lines, and 22.7 °C for
‘Vlaspik’ from 8 to 17 Aug. (LSD = 1.2, P S 0.05).

For the ﬁrst to third harvests, yields of ‘Vlaspik’ and commercial small types
were very similar, whereas the two reduced branching types had signiﬁcantly lower yield;
however, the disease occurrence at O and 4 DPH was not signiﬁcantly different among
the architecture types (Figure 2-4A-C). The compact lines, determinate with no-laterals
(PI 466921), and the rest of the reduced branching types could only be harvested at the
third and fourth harvests. At 4 DPH, most of the architectural types showed increased
disease occurrence compared 0 DPH; however, compact line PI 308916 remained
signiﬁcantly lower at both 0 and 4 DPH (Table 2-4). The difference in disease
occurrence did not result from fruit resistance per se, as direct inoculation of the PI
308916 fruit showed the fruit to be comparably susceptible to ‘Vlaspik’ (Table 2-4).
Interestingly, many fruit of PI 308916 were held above the ground throughout the

experiment (Figure 2-5).

47

 

50

4o-

30~

 

20*

Fruit (nonIot)

10~

 

 

 

 

 

  
    

30 ‘ —O— Vlaspik
-~-O-- Commercial
-1— Reduced

  

20~

10‘

 

 

100 -

80‘

60-

40‘

20‘

 

 

Diseased fruit at 4 DPH (%) Diseased fruit at 0 DPH (%)

 

 

Harvest

Figure 2-4. Mean fruit munber per plot (A) and percent disease occurrence at harvest (B)
and four days post harvest (C) for ‘Vlaspik’; mean of the commercial lines ‘Arabian’,
‘Colt’, ‘Palamino’, and ‘Stallion’; and reduced branching types (mean of PI 192940,
227207, and 401734). Each data point is the mean of 3 - 16 plots (3 replicate plots per

genotype) 1 SE.

48

 

FigureZ-S. Fruit position for PI 308916 early (A) and late (B) in the season.

49

Discussion

Developing cucumber ﬁ'uit are located under the canopy where warm and moist
environmental conditions are optimal for germination and grth of P. capsici
(Hausbeck and Lamour, 2004) Therefore, we sought to determine whether using
aitemate plant architecture which has less canopy coverage over the fruit and allows for
increased air circulation, or that removes fruit from direct contact with the soil, might be
an effective strategy to reduce disease severity. A trellis study was conducted as an
extreme test of this hypothesis. There were differences in the time of canopy ﬁlling and
temperature under the canopy among the narrow-spaced, wide-spaced and trellis plots.
As was predicted, the narrow plots had the highest temperatures under the canopy where
ﬁ'uit were located. The wide spaced plots had generally intermediate temperatures, and
the trellis plots had lowest temperatures, likely due to increased air circulation. Although
we were not able to measure humidity directly, it is likely that the more open conditions
around the trellised fruit also resulted in reduced relative humidity. Even though there
was open ground between rows within the wide spaced plots, the fruit were generally
located under a dense canopy of the parent plant and exhibited a similar percent infection
as narrow spaced plots.

The trellis plots however, had signiﬁcantly lower rates of P. capsici infection
suggesting that canopy conditions and/or removing the fruits from the ground can help
reduce disease incidence. Although trellis production is not economically feasible for
pickling cucumber, these results suggest that alternate cucumber architecture types which

simulate trellis conditions might contribute to disease control strategies.

50

The effect of architectural differences on canopy development was examined
using near-isogenic lines differing for leaf size and determinacy. Comparison of
temperature under the canopy and other types showed that the standard type
(indeterminate and standard leaf) had signiﬁcantly higher daily maximum temperature
than the alternate architectural types (little leaf, determinate, and compact). Goode et al.
(1980) suggested that canopy differences in little leaf plots contribute to reduced
occurrence of the soil-bome R. solani and Pythium apham'dermatum (Edson) Fitzp.
diseases. In our study, the standard architecture lines had signiﬁcantly higher occurrence
of P. capsici-induced fruit rot than the little leaf, compact or determinate types suggesting
an effect of architecture; however, disease occurrence in the severe late-season conditions
still exceeded economically feasible levels.

A representative example of the cucumber gennplasm was screened for other
architectural variants that might be useful for reducing occurrence of P. capsici infection.
There was a range of variation for architectural traits with up to 10-fold differences in
various traits. Comparison of architectural traits showed that stem length is correlated
with intemode length as would be expected, although lack of perfect correlation can also
reﬂect differences in growth pattern inﬂuencing other features that can affect vine length
such as fruit and ﬂower location, branching, and determinacy. The lack of correlation of
several architectural traits indicates that different phenotypes appear to be inherited
separately, and so could be genetically manipulated independently.

Possible variants which might be helpful in reducing P. capsici disease severity,
included reduced branched types which produced a more open canopy, and compact

types with initial upright fruit position that might reduce fruit contact with the soil. When

51

the reduced branching PIs were tested at commercial planting densities, they exhibited
increased main stem length and branching number compared to the initial screening
which was done with wide spacing. Schultheis et al. (1998) did not observe an effect of
plant spacing on cucumber vine length. The observed differences in length may be due to
different physiological age of the plants at the time of measurement or different grth
conditions in the two seasons. Determinate and compact vines appear to be less affected
by environmental conditions. The increase in branch number might be due to changes in
planting density, since Horst and Lower (1977) noted that plant density can affect lateral
number.

Of the architectural variants tested, reduction in disease occurrence was observed
for only one, PI 308916. Direct fruit inoculation tests showed that the reduced infection
of PI 30896 was not due to fruit resistance; therefore, the low disease incidence in P1
308916 may result from its unique architecture which allows for many of the fruit to be
held above the ground. This trait may be useful for future strategies to reduce losses due
to P. capsici infection.

In summary, alternate plant architecture could be helpful in reducing P. capsici
infection on pickling cucumber. Varying architecture resulted in modiﬁed canopy
structure as observed visually and by canopy temperature. However, disease incidence
data suggest that canopy conditions are less important for disease control than removal of
fruit from the soil. The trellis study and plots with the compact PI 308916, suggest that
reducing contact of young cucumber fruit with the soil can be a means to alleviate P.

capsici disease severity.

52

Acknowledgments

We thank the Pickle Seed Research Fund and MSU-GREEEN for support of this
project. We thank Dr. Jack Staub, the Plant Introduction Station (Ames, Iowa), and
Seminis Vegetable Seed Inc. for providing seed. We thank Steve Suzura and Dr. Mary
Hausbeck and her lab members, R. Bounds, B. Cortright, A. Gevens, and B. Harlan, and
for their help in ﬁeld and fruit screening experiments and Bill Chase, Gary Winchell, and
Ron Gnagney for farm assistance. We also thank Drs. Jim Kelly and Mary Hausbeck for

their helpful reviews of the manuscript.

53

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Coyne, D. P. 1980. Modiﬁcation of plant architecture and crop yield by breeding.
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Coyne, D. P., J. R. Steadman, and F. N. Anderson. 1974. Effect of modiﬁed plant
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mold severity, and components of yield. Plant Dis. Rept. 58:379-3 82.

Erwin, D. C. and O. K. Ribeiro. 1996. Phytophthora capsici. P. 262-268. In:
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MN.

Fuller, P. A., J. R. Steadman, and D. P. Coyne. 1984. Enhancement of white mold
avoidance and yield in dry beans by canopy elevation. HortScience 19:78-79.

George Jr., W. L. 1970. Genetic and environmental modiﬁcation of determinate plant
habit in cucumbers. J. Amer. Soc. Hort. Sci. 95:583-386.

Gevens A. J ., K. Ando, K. Lamour, R. Grumet, and M. K. Hausbeck. 2006. Development

of a detached cucumber fruit assay to screen for resistance and effect of fruit age
on susceptibility to infection by Phytophthora capsici. Plant Dis. 90:1276-1282.

Goode, M. J ., J. L. Bowers, and A. Bassi Jr. 1980. Little-leaf, a new kind of pickling
cucumber plant. Arkansas Farm Research 29: 4.

Grumet, R. and R. Duvall. 1993. Testing the effect of the determinate shoot grth allele
on cucumber root growth. HortScience 28:847-849.

Halterlein, A. J ., G. L. Sciumbato, and W. L. Barrentine. 1981. Use of plant desiccants to
control cucumber ﬁ'uit rot. HortScience 16:189-190.

Hanna, H. Y., A. J. Adams, and R. N. Story. 1987. Increased yield in slicing cucumbers
with vertical training of plants and reduced plant spacing. HortScience 22232-34.

Hausbeck M. and K. Lamour. 2004. Phytophthora capsici on vegetable crops: research
progress and management challenges. Plant Dis. 88:1292-1302.

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Horst E. K. and R. L. Lower. 1977. Effect of plant density on lateral number and main
stem length in Cucumis sativus L. and Cucumis hardwickii. HortScience 12:23 5-
235.

Kauffman, C. S. and R. L. Lower. 1976. Inheritance of an extreme dwarf plant type in the
cucumber. J. Amer. Soc. Hort. Sci. 101:150-151.

Knerr, L. D., J. E Staub, D. J. Holder, and B. P. May. 1989. Genetic diversity in Cucumis
sativus L. assessed by variation at 18 allozyme coding loci. Theor. Appl. Genet.
78:119-128.

Kolkman, J. M. and J. D. Kelly. 2002. Agronomic traits affecting resistance to white
mold in common bean. Crop Sci. 42:693-699.

Konsler, T. R. and D. L. Strider. 1973. The response of cucumber to trellis vs. ground
culture. HortScience 8:220-221.

Kreutzer, WA. 1937. A Phytophthora rot of cucumber fruit. Phytopathology 27:955.

Lamour, K. H. and M. K. Hausbeck. 2000. Mefenozam insensitivity and the sexual stage
of Phytophthora capsici in Michigan cucurbit ﬁelds. Phytopathology 902396-400.

Lamour, K. H. and M. K. Hausbeck. 2001a. Investigating the spatiotemporal genetic
structure of Phytophthora capsici in Michigan. Phytopathology 91:973-980.

Lamour, K. H. and M. K. Hausbeck. 2001b. The dynamics of mefenoxarn insensitivity in
a recombining population of Phytophthora capsici characterized with ampliﬁed
fragment length polymorphism markers. Phytopathology 91:553-557.

Lamour, K. H. and M. K. Hausbeck. 2002. The spatiotemporal genetic structure of
Phytophthora capsici in Michigan and implications for disease management.
Phytopathology 92 :68 1 -6 84.

Lamour, K.H. and M. K. Hausbeck.2003. Effect of crop rotation on the survival of
Phytophthora capsici in Michigan. Plant Dis. 87:841-845.

Leonin, L.H. 1922. Stern and fruit blight of peppers caused by Phytophthora capsici sp.
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Park, S. J. 1993. Response of bush and upright plant type selections to white mold and
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Plant Variety Protection Certiﬁcate. 1993. H-19 cucumber. PVPO no. 8900073. US Dept.
Agriculture, Agriculture Marketing Service. Washington, DC.

Russo, V. M., B. W. Roberts, and R. J. Schatzer. 1991. Feasibility of trellised cucumber
production. HortScience 26: 1 156-1 158.

Schultheis, J. R., T. C. Wehner, and S. A. Walters. 1998. Optimum planting density and
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J. Plant Sci. 78:333-340.

Serce, S., J. P. Navazio, A. F. Gokce, and J. E. Staub. 1999. Nearly isogenic cucumber
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56

Chapter 3

Age related resistance in cucumber and various cucurbit fruit to infection by

Phytophthora capsici

Abstract

Cucumber fruit rot caused by Phytophthora capsici causes severe losses in
vegetable production, including many cucurbit crops. In the course of screening for
genetic sources of resistance among cucumber germplasm, fruit age/size appeared to be a
factor in susceptibility to infection by P. capsici. To verify this observation, greenhouse
grown ﬁ'uits of known age were inoculated with P. capsici. Cucumber fruits were most
susceptible to P. capsici when they were very young and rapidly elongating, but then
developed age-related resistance (ARR) as they approach full length at 10-12 days post
pollination (DPP). This was observed in both the greenhouse and ﬁeld, and for several
genotypes. Seven additional cucurbit crops, zucchini, yellow summer squash, acorn
squash, pumpkin, butternut squash, melon, and watermelon, representing four species
(Cucurbita pepo, C ucurbita mochata, C ucumis melo, and C itrullus lanatus) also
exhibited size—related decrease in susceptibility, but to varying degrees. In the ﬁeld,
infection primarily occurs at the blossom end. To determine whether preferential
infection of the blossom end was due to position-related differences, or was a result of
earlier contact with inocultun-containing soil, greenhouse-grown cucumber fruit of
known age were inoculated with P. capsici at the peduncle and blossom ends. The
youngest fruits supported sporulation on both ends. As fruit age increased, the peduncle

end became less susceptible sooner, suggesting a developmental gradient within the fruit

57

inﬂuencing susceptibility. This difference in susceptibility was signiﬁcant for cucumber,
zucchini, acorn, and butternut squash fruits. To examine whether the basis of ARR in
cucumber fruit-P. capsici is due to changes in surface or mesocarp properties, exocarp
sections from 8 DPP (susceptible) or 15 DPP (resistant) ﬁ'uit were placed onto intact 8 or
15 DPP fruit, inoculated with P. capsici zoospore suspensions and examined for disease
development. All exocarp sections from 8 DPP supported sporulation, but not 15 DPP,
indicating that the exocarp plays a signiﬁcant role in the transition to resistance.
Microscopic analysis of zoospore inoculated fruit surface showed that short germ tubes
and appressorium formation was signiﬁcantly higher on 8 DPP fruit while longer and

aberrant germ tubes are more prevalent on 16 DPP fruit.

Introduction

In recent years, Phytophthora capsici has become an increasingly severe pathogen
causing disease on a wide range of vegetable crops, including cucurbit crops, where it can
cause devastating yield losses (Babadoost, 2004; Hausbeck and Lamour, 2004). In some
cases, losses of up to 100 % have been reported and growers have had to abandon
production ﬁelds (Babadoost, 2004). Phytophthora capsici is a soil-home Oomycete
pathogen that can cause diseases in many plant organs and at various growth stages of the
host, e.g., damping-off on processing pumpkin, and fruit rot on cucumber. It is favored
by wet and warm environments and can spread with water such as rain splash or
irrigation. The symptoms observed during the disease progression of P. capsici infected
fruit is a depressed fruit surface with a water soaked appearance followed by white

cottony mycelium covering the affected region.

58

Pickling cucumber crops in Michigan have experienced major losses to P. capsici
in recent years (Hausbeck and Lamour, 2004). Field observations show that cucumber
fruit are susceptible to P. capsici, while roots, vines, and leaves are much less
susceptible. Fields will frequently look healthy at harvest time as judged by vegetative
growth, but the fruits can be heavily diseased (Hausbeck and Lamour, 2004). Cucumber
fruit are usually located under the canopy where the environment is moist, warm, and
close to the pathogen in the soil, thus favoring conditions for disease development.
Trellis and plant architecture studies have indicated that removal of fruit from the soil
surface can reduce disease incidence (Chapter 2; Ando and Grumet 2006).

Control of the disease by chemical and cultural practices remains a challenge.
Resistance to the common fungicide, mefenoxam, has emerged (Lamour and Hausbeck,
2000). Rotation to non-host crop is of limited value, since oospores of P. capcisi can
survive in the soil for a long period of time, up to 10 years (Hausbeck and Lamour,
2004). Wider plant spacing, cover crops, trellises, or variant architectural phenotypes
may help to control the disease by facilitating better fungicide coverage, modifying the
microclimate, or reducing contact with the inoculum-containing soil, thereby facilitating
disease avoidance (Ando and Grumet, 2006; Ngouajio et al., 2006; Ristaino et al., 1997;
Wang and Ngouajio, 2008). However, these methods can be difﬁcult to implement in a
commercial setting. The most desirable solution would be to ﬁnd resistant cultivars, but
this has met with mixed success. Efforts are underway to identify sources of resistance in
C ucurbita pepo (Padley et al., 2008) and to introgress resistance from the wild C ucurbita
species, C. lundelliana and C. okeechobeenesis, into C. mocshata (Padley et al., 2009).

To our knowledge there have not been studies to identify sources of resistance to P.

59

capsici in watermelon or melon. Screening of a diverse collection of cucumber
germplasm accessions for fruit resistance to P. capsici has not identiﬁed any resistant
genotypes to date (Gevens et al., 2006).

In the course of screening for sources of P. capsici resistance in cucumber
germplasm, ﬁ'uit used as susceptible controls did not become uniformly infected. Prior
observation suggested that susceptibility of the fruit might be related to fruit size or age;
the smaller fruit appeared to be more susceptible than larger fruit. Age-related resistance
(ARR), also known as adult plant resistance or developmentally related resistance, in
which an older plant or plant organ (e. g., leaf or fruit) becomes less susceptible to
pathogens, has been observed in many plant-pathogen systems, including disease caused
by fungi, oomycetes, bacteria, and viruses, and is becoming increasingly recognized as an
important component of plant defense against infection (Develey-Riviere and Galiana,
2007; Panter and Jones, 2002; Whallen, 2005). If ARR is present in the cucumber fruit-
P. capsici interaction, it would affect the methods used for accurate screening. It would
also have implications for which stage of fruit is likely to become infected, possible
management implications with respect to spray practices, and insight into approaches to
develop more resistant ﬁ'uit.

Further examination of P. capsici infected cucumber fruit in the ﬁeld led to the
observation that ﬁeld-grown cucumbers are typically covered with mycelium on the
blossom end of the fruit rather than the peduncle end. This observation could be either
due to the tendency of the blossom end to come into contact with the inoculum
containing soil sooner than the stem end, or that there is a susceptibility gradation within

the fruit such that the blossom end is more susceptible than the stem end.

60

In this chapter, I characterized the effect of fruit age on susceptibility to P. capsici
in cucumber and other cucurbit crops representing four species: melon (Cucumis melo),
watermelon (Citrullus lanatus), buttemut squash (C ucurbita mocshata), and four
C ucurbita pepo crops, zucchini, yellow summer squash, acorn squash, and pumpkin. I
also examined the basis for age-related increased resistance by asking whether it is due to
changes in surface or mesocarp properties, and determined the nature of the difference in

occurrence of disease at the blossom and peduncle end in cucumber and other cucurbits.

Materials and methods

Cucumber screening for P. capsici resistance

A set of 150 cucumber accessions was supplied by the North Central Regional
Plant Introduction Station, Ames, IA. The ﬁrst 100 genotypes tested were selected based
on isozyme data to serve as a representative sample of the cucumber gennplasm with
maximum genetic variance (Knerr et al., 1989). Another 50 genotypes that had been
selected based on possible architectural variation were also included in the screening
(Ando and Grumet, 2006). Seeds were sown in the Michigan State University (MSU)
Research greenhouse, East Lansing, MI and 14-day—old seedlings were transplanted to
the MSU Horticulture Research and Teaching Center (HRTC) in a nonreplicated trial on
a ﬁeld with no history of P. capsici infestation in summer 2003. Seedlings were planted
0.46 m apart in 3.67 m single-row plots with 1.52 m between rows. Plastic mulch was

used to control weeds and local standard commercial production guidelines were

61

followed for insect control (Bird et al., 2005). Irrigation was provided by rain or
overhead irrigation to 25 mm a week.

Harvested fruit from commercial standard (‘Vlaspik,’ Seminis Vegetable Seeds,
Oxnard, CA) and test genotypes were inoculated with mycelial/sporangial plugs of P.
capsici isolate OP97 (an isolate of P. capsici, provided by Dr. Mary Hausbeck Lab at
Michigan State University) without wounding according to the method of Gevens et al.
(2006). Brieﬂy, a 7 mm agar plug containing 7-11 day old P. capsici mycelia was
covered with a sterile microcentrifuge tube secured to fruit with petroleum jelly and
incubated at room temperature under ﬂuorescent lighting. Four to eight fruit were tested
for each genotype. Initial fruit examination was made 4 days post inoculation (DPI).
Symptom development was monitored and scored as 1 — no symptoms, 2 — water
soaking, and 3 — sporulation. Fruit with no symptoms at 4 DPI were monitored daily and
maintained for further observation for a minimum of 10 DPI, or until the occurrence of
water-soaked lesions and sporulation.

The PIs identiﬁed in 2003 for which less than 50 % of the tested fruit exhibited
pathogen sporulation at 10 DPI (PIs: 24956] Thailand, 255937 Netherlands, 263047
Russia, 288990 Hungary, and 304805 US.) were reevaluated using greenhouse-grown,
hand-pollinated fruit in 2004. Seedlings were planted in 3.8-liter pots with sterile media
(BACCTO, Michigan Peat Co., Houston, TX) under supplemental lighting (18/6 h light
/dark) at 21-25 °C, fertilized with 300 mg-L'l nitrogen from Peters Professional® 20-
20-20 General Purpose water soluble fertilizer (The Scotts Company LLC, Marysville,
OH) once a week. Pest control was performed according to the standard management

practices in the greenhouse. ‘Vlaspik’ was used as a commercial standard. A minimum

62

of ﬁve fruit from each genotype were harvested at 7 and 14 days post pollination (DPP),
and inoculated as described above. Fruit were observed initially at 4 DPI, then
maintained for further evaluation until 10 DP] and scored for water-soaked lesions and
pathogen sporulation.
Effect of fruit age

Cucumber cultivar ‘Vlaspik’, was grown in a research greenhouse at MSU during
spring 2004 as described above. Flowers were hand-pollinated at 2 to 3 day intervals to
ensure a range of fruit ages from 2 to 18 DPP at the day of harvest. Harvested fruit were
washed with distilled water, air-dried, and measured for fruit length and diameter. The
varying-aged fruit were placed at random in aluminum trays and inoculated with P.
capsici and scored for symptoms as described above. The experiment was repeated three
times with approximately 15-20 ﬁ'uit per experiment.

Fruit age experiments were also conducted using fruit from ﬁeld-grown plants.
The slicing cucumber cultivar ‘Straight Eight’ was grown at the MSU HRTC in ﬁelds
with no history of P. capsici during the summer of 2004 using standard management
practices. Cucumber fruit representing a range of sizes were harvested on four dates; 15
to 20 fruit were tested per harvest date and fruit with visible wounds were not used. Fruit
were prepared and inoculated as described above.
Parthenocarpic fruit experiment
Parthenocarpicly developed ‘Vlaspik’ fruits were grown in the MSU Research
greenhouse during spring 2006 as described above. Fruits were harvested, inoculated, and

scored as described above. At 10DPI, fruit were cut longitudinally to search for the

63

presence of seeds; data from the fruits with seeds were excluded. This experiment was
repeated 3 times with a total of 58 fruit.
Fruit growth analysis

Cucumber ‘Vlaspik’ plants were grown in the greenhouse during the summer of
2007 under the same conditions described earlier. Fruit were marked with waterproof
India ink (Sanford LP, Oak Brook, IL) with equally spaced 5 dots along the fruit length.
Length between the dots which include length between the both ends and the neighboring
dots were measured daily up to 34 DPP.
Fruit position experiments

‘Vlaspik’ fruits were grown in the greenhouse during the spring of 2004 under
the same conditions as described earlier. Flowers were hand-pollinated over a period of
days to allow for simultaneous harvest of fruit ranging in age from 6 to 14 DPP.
Inoculation and disease screening at 4 DPI was as described earlier, except that two 6 mm
diameter plugs of P. capsici isolate OP97 were placed approximately 2 cm from the
peduncle- and blossom-end of each fruit.
Exocarp transfer experiment with zoospore

Zoospore production and inoculation preparation was performed according to
Gevens et al. (2006). Concentration of the zoospore suspensions was determined with a
hemacytometer and diluted to 1 x 106 per ml. Exocarp sections (3 cm x 3 cm x 1-2 mm)
ﬁ'om the middle part of the fruit were excised from both 8 and 15 DPP fruit with petit
knife or razor blade without introducing nicks. A sterile plastic tube (0.6 cm height, 0.8
cm diameter) was placed on the exocarp section and anchored to underlying intact 8 or 16

DPP fruit using a strip of 1 cm wide paraﬁlm. A twenty-two gage sterile needle was used

64

to deliver 30 ul zoospore suspension into the tube by penetrating the paraﬁlm; the needles
did not come in contact with the fruit tissue. Intact 8 and 15 DPP fruits were similarly
inoculated and included in each tray as control. Inoculation and scoring of symptom
development was as described above.
Eﬁ'ect of fruit surface on zoospore germination

Ten greenhouse-grown cucumber fruit were harvested at 8 and 16 DPP and were
inoculated with 30 u] P. capsici zoospore suspensions at concentration of l x 104, as
described earlier. After 24 hours of inoculation, fruits were removed from the trays and
the zoospore suspensions were allowed to air dry. A thin layer of clear nail polish
(Markwins Beauty Products, Inc., City of Industry, CA) was applied on the inoculated
surface of the fruits or equivalent location for non-inoculated control fruits as was
described in Iwaro et al. (1997). After hardening, the nail polish was removed and
mounted on the cover glass for microscope analysis. Each sample was divided into three
sub-sections and twenty zoospores were counted randomly within each sub-section.
Zoospores were examined for germination, length of germ tube, (short — less than 1x
zoospore cyst diameter, medium — 1-5x diameter, long - greater than 5x diameter),
presence of appressoria, and occurrence of aberrant germ tubes. Fruits were scored for
symptom development at 6 DPI as described earlier.
Additional cucurbit fruit screening

Cucmnber ‘Vlaspik’; muskmelon ‘Odyssey’ (Rispens Seeds, Inc., Beecher, IL);
butternut squash ‘Waltham’(Seedway LLC., Hall, NY); watermelon ‘Crimson Sweet’
(Seedway); zucchini ‘Black Beauty’(Seedway); yellow summer squash ‘Horn of

Plenty’(Seedway); acorn squash ‘Royal Acorn’ (Seeds of Change, Santa Fe, NM); and

65

pumpkin ‘Baby Pam’ (Seedway), were grown in a ﬁeld with no history of P. capsici
infestation at the MSU HTRC during the summer of 2006. Crops were planted in 15 m
rows, 3.3 m between rows, and 50 cm spacing within rows, except cucumber which was
planted at 4 cm spacing within row. Commercial pest control was applied as described
earlier. Plastic mulch was used to suppress weeds. Irrigation was provided by rain or
trickle irrigation to provide 25 mm per week.

Fruits were harvested on at least two dates for each crop to provide a total of 45-
93 ﬁ'uits per crop, representing a range of stages of development as assessed by ﬁ'uit size
and appearance. Harvested ﬁ'uits were washed with distilled water, air-dried, and
measured for length and width. The ﬁ'uits were then inoculated on both ends with P.
capsici as described earlier.

Greenhouse studies were also performed for acorn squash ‘Autumn Delight’
(Siegers Seeds Co., Holland, MI) butternut squash ‘Waltham’, and pumpkin ‘Baby Pam’
in the MSU Research Greenhouse during the spring and summer of 2008. Seeds were
sown into 38 L plastic pots ﬁlled with BACCTO media and plants were grown under the
same environmental conditions as described earlier. Flowers were hand-pollinated daily
over a 45 day period to provide a set of fruit of known, different ages that could be
harvested and tested simultaneously. The experiments were repeated at least twice for
each cr0p with sixteen plants per crop per experiment to provide a total of 67-79 fruits
per crop. Harvested fruits were measured for length and width, examined for color and
external surface properties, then processed for inoculation on both ends and scored for

symptoms as described above.

66

Statistical analysis

Data were analyzed by ANOVA using the SAS program 9.1 (SAS Institute Inc.,
Cary, NC) with mixed procedures; when appropriate, LSD was used for multiple
comparisons. Regression analysis was performed using SigmaPlot 8.02 (SPSS Inc.,
Chicago, IL). Blossom/peduncle end comparisons of the same fruit were performed by
using chi-square contingency analysis on two trials of the same subject according to

Steele and Torrey (1960).

Results
Cucumber fruit exhibit age-related resistance to P. capsici

The majority of cucumber genotypes screened for resistance to P. capsici were
rapidly infected and exhibited sporulation by 5 DPI (Fig 3-1A). When the evaluation
was extended to 10 DPI, infection levels increased, with ca. 90 % of the P15 exhibiting
sporulation on >50 % of the fruit tested (Figure 3—1A). When accessions exhibiting a low
percentage of fruit with pathogen sporulation were rescreened in 2004, all were as
susceptible as the commercial ‘Vlaspik’ standard (data not shown).

Symptom development on ‘Vlaspik’ fruit that were included as susceptible
controls in each tray of the germplasm was inconsistent. Smaller, younger fruit tended to
be more readily infected, while larger, older fruit were less frequently infected. To
directly test the effect of fruit age, sets of greenhouse-grown, hand-pollinated fruits of
known ages, ranging from 2 to 18 DPP were inoculated at the same time (Figure 3-2A-
C). At 4 DPI, most fruit younger than 10 DPP exhibited sporulation, whereas after 12

DPP, most fruit remained symptom-free. Fruit of intermediate ages (10 to 12 DPP) often

67

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Figure 3-1. A. Frequency distribution showing number of cucumber genotypes for which
sampled fruit showed the indicated sporulation (%) at 5 (blank bar) and 10 (striped bar)
days post inoculation (DPI). Asterisks (*) indicate ‘Vlaspik’ value. B. Disease response
of the candidate genotypes identiﬁed in cucumber screen 2 on greenhouse-grown hand-
pollinated fruit harvested at 7 and 14 days post pollination (DPP). Fruit symptoms were
scored for disease response at 4 DPI as: O-no symptoms, l-water soaking, and 2-
sporulated. "' 7 and 14 DPP fruit within a genotype were signiﬁcantly different from each
other, t-test (P3005). Each value is the mean of minimum 5 fruit i SE.

68

exhibited water-soaked regions without sporulation. Symptom-free fruit were usually
oversized for pickling cucumbers (length >14 cm, width >4.5 cm).

The transition from susceptible to more resistant appeared to coincide with the
transition away from the period of rapid fruit elongation (approximately 12 cm length),
with a sharp decline in disease rating at approximately 10-12 DPP (Figure 3-2A, D and
E). F ield-grown cucumbers (‘Straight Eight’) harvested to include a range of fruit sizes
from 2 to 22 cm in length, were also tested for response to P. capsici inoculation.

Although growth rate and size can be inﬂuenced by environmental conditions, the
relationship between fruit size and disease susceptibility followed the same trend as
observed with greenhouse grown fruit (Figure 3-2F).

Fruit age effect was further examined with the candidate genotypes identiﬁed in
the germplasm screen using both 7 and 14 DPP fruit to target pre- and post-transitional
stages based on the ‘Vlaspik’ results. At 7 DPP, the majority of ﬁ'uit from all genotypes
supported sporulation; the remainder developed water-soaked regions (Fig 3-1B). At 14
DPP, fruit from three of the ﬁve candidate genotypes (Pls: 263047, 288990, and 304805)
were signiﬁcantly less susceptible than at 7 DPP (t-test P S 0.05), with most of these fruit
showing water-soaking symptoms or no symptoms at all rather than sporulation. In no
case were older ﬁ'uit more susceptible than younger fruit.

Preliminary characterization of the age-related resistance in cucumber
Effect of seed formation
The time period from 12-16 DPP is associated with noticeable changes in seed

development including hard seed coat formation (Chapter 4). To test whether seed

69

Figure 3-2. Relationship between cucumber fruit age and size, and response to
inoculation with Phytophthora capsici. A-E. Response of greenhouse-grown hand
pollinated fruit. Greenhouse ﬁ'uit age experiments were repeated three times with
approximately 15 to 20 fruit per experiment. The same trends were observed in each
experiment. Data presented are pooled from the three experiments. A, Response of
greenhouse-grown, hand-pollinated ‘Vlaspik’ fruit to P. capsici inoculation in relation to
fruit age and length. B, Response of ‘Vlaspik’ fruit of various ages to inoculation with P.
capsici. C. Mean disease rating (:1: SE) of fruit harvested at 3 to 18 days post pollination
(DPP). Rating scale: 0 — no symptoms, 1 — water-soaking, and 2 - sporulated; eachpoint
(with the exception of 18 DPP) is the mean of 3 to 12 fruits. D. Mean disease rating (i
SE) of cucumber fruit in relation to fruit diameter . Each point is the mean of 4 to 10
fruits. E. Disease response in relation to fruit diameter and length. F. Disease response
of inoculated ﬁeld-grown ‘Straight Eight’ fruit in relation to fruit diameter and length.
Symptom data were taken at 4 days post inoculation (DPI). Data are pooled from fruit
sampled on four dates. r2 values refer to quadratic binomial indicating a change in the rate
of growth over time.

70

Disease rating at 4 DPI

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71

development is related to the age-related resistance, parthenocarpic fruit were tested for
susceptibility to infection by P. capsici. Various sizes of parthenocarpically developed
greenhouse grown fruits also showed changes in susceptibility with fruit development
(Figure 3-3A). Fruit at 1cm diameter developed either water soaking or sporulation
(mean disease ratingiSE, 2.6i0.1), whereas fruit with diameters greater than 3 cm did
not sporulate (1.6i0.05). Disease rating of non-parthenocarpic, ﬁeld grown ﬁ'uits also
decreased with increased with diameter, similar to parthenocarpic fruit (Figure 3-3 B).
Role of fruit surface

Preliminary tests were conducted to examine whether the age-related decrease in
susceptibility is associated with the fruit surface (exocarp), mesocarp or both. Fruits
peeled prior to inoculation all formed sporulating lesions, whereas intact control fruit did
not become infected at 4 DPI (data not shown), suggesting that the fruit surface is an
important factor for the resistance of older fruits to infection by P. capsici. However,
these experiments do not allow us to rule out possible effects of wounding due to peeling.

To further study the role of the exocarp using intact (non-wounded) fruit, exocarp
exchanges were made between fruits at 8 and 15 DPP (i.e., exocarp piece from one fruit
were placed on top of a second, intact fruit and inoculated with zoospores) (Figure 3-4A).
The 8 DPP fruit surface pieces developed either water-soaking or sporulation regardless
of ﬁ'uit age underneath (mean disease ratingiSE: 2.6:i:0.2), as did intact fruit (Figure 3-
4B). Fruit surface sections from 15 DPP fruit, like intact 15 DPP ﬁ'uit, generally did not
sporulate regardless of fruit age underneath; however, some developed water-soaked
symptoms (1 .3:I:0.2). The fruit underneath the 8 DPP ﬁ'uit surface pieces showed

susceptibility similar to the fruit age study; 8 DPP fruit generally sporulated while 15

72

 

 

 

 

 

 

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Figure 3-3. A, B. Mean disease rating at 4 days post inoculation (DPI)of parthenocarpic
(A) and ﬁeld-grown non-parthenocarpic (B) cucumber fruit in relation to fruit length to
inoculation by Phytophthora capsici. Disease rating scale: 1 — no symptom, 2 — water
soaking, and 3 — sporulation. Each point is the mean of at least 5 fruit :tSE.

73

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Peel and fruit age

Figure 3-4. A. Picture of 8 and 15 days post pollination (DPP) exocarp section and intact
fruit underneath to infection by Phytophthora capsici zoospore suspension. B. Disease
rating at 4 days post inoculation (DPI). Disease rating: 1 — no symptoms, 2 — water
soaking, and 3 — sporulation. Left bars are the exocarp section and right bars are fruit
underneath. Each bar is mean of at least 9 fruit and :tSE.

74

DPP fruit generally did not sporulate even in the presence of sporulating 8 DPP fruit
surface sections. However, when 15 DPP ﬁ'uit surface pieces were inoculated, the
underlying 8 DPP fruit did not sporulate, indicating that the 15 DPP fruit surface sections
protected the underlying 8 DPP ﬁ'uit. These results suggest that 15 DPP fruit surface
section possesses properties that inhibit P. capsici infection. Equivalent were obtained
with mycelium inoculation (data not shown).
Effect of fruit surface on zoospore germination

Since the exocarp is the ﬁrst point of contact of the inoculum, it is possible that
exocarp properties inﬂuence P. capsici development in pre-infection stage. Although, as
before, the young ﬁ'uit were infected while older fruit were resistant (Figure 3-5A),
preliminary results showed no difference in percent zoospore germination when placed
on 8 or 16 DPP fruits (Figure 3-5 B). However, short gerrn-tubes were more prevalent on
8 DPP fruit, while medium and long germ tubes were more frequent on 16 DPP fruit.
Appressoria formation was signiﬁcantly higher on 8 DPP than 16 DPP fruit, whereas
aberrant germ tubes were signiﬁcantly higher on 16 DPP fruit.

Effect of fruit position

P. capsici infected cucumber ﬁ'uit in the ﬁeld are typically covered with
mycelium on the blossom end of the ﬁ'uit rather than the stem end (Figure 3-6A). To
determine whether this phenomena is due to greater tendency of the blossom end of the
fruit to touch the soil, or if there is a difference in susceptibility due to position on the
fruit, hand-pollinated greenhouse grown fruit (ranging 6 to 14 DPP) were inoculated with
P. capsici mycelium approximately 2 cm from the blossom end and the peduncle end

(Figure 3-6B). When fruit were very young (6 DPP), both the blossom end and the

75

 

 

 

 

 

 

 
  
  

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Figure 3-5. A. Mean disease rating of 8 and 16 days post pollination (DPP) to infection
by Phytophthora capsici zoospore suspension at 6 days post inoculation (DPI). Disease

rating: 1 — no symptoms, 2 — water soaking, and 3 — sporulation. B. Zoospore response

on the surface of 8 and 16 DPP cucumber fruits. Each value is the mean i SE of 10 fruit,
3 subsamples/fruit, 20 zoospores/subsample. Germ tube length: short <lx zoospore cyst
diameter; medium 1-5x diameter; long >5x diameter.

76

Figure 3-6. A. Typical symptoms of Phytophthora capsici in the ﬁeld - mycelium cover
the blossom end of fruits. B, Mycelium inoculation of the peduncle and blossom ends of
6 to 14 days post pollination (DPP) fruit. The picture was taken at 4 days post
inoculation (DPI). C. Equally spaced ﬁve dots (1 — peduncle end, 5 — blossom end) were
drawn on the surface of each fruit to measure grth from various parts of fruit. D.
Symptoms of ﬁ'uit ranging from 6 to 14 DPP were evaluated at 4 DPI (0 — no symptom, 1
— water soaking, and 2 — sporulation). Each point is the mean of a minimum of 10 fruits
i SE. B. Fruit growth from peduncle (1-2) and blossom end (4-5) which were measured
daily up to 34 DPP. Each point is a mean of 4-10 fruits.

77

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peduncle end sporulated (Figure 3-6D). However, as fruit develop, the peduncle end
becomes less susceptible earlier than the blossom end, so that sporulation occurs on the
blossom end, but not the peduncle end (e. g. at 8-12 DPP). At 14 DPP the peduncle end
did not develop symptoms whereas the blossom end sometimes developed water soaked
regions. Growth analysis of developing fruit showed that the peduncle end stopped
elongating earlier (10-12 DPP) than the blossom end (14-16 DPP) (Figure 3-6C, E).
Age-related resistance of fruit from other cucurbit species to P. capsici

Additional types of cucurbit fruit were tested to see if they also exhibit age related
resistance. The fruit of different cucurbit crops exhibited variability for overall
susceptibility (Figure 3-7). When fruit of all sizes were grouped together, summer
squash, zucchini and melon were the most susceptible with the greatest percent producing
sporulating lesions at 4 and 10 DPI and the shortest time to 50 % sporulating fruit (Figure
3-7D-F). Both summer squash and zucchini were also notable for rapid development of
water soaking symptoms; more that 50 % of the tested fruit exhibited water soaking by 1
DPI (Figure 3-7A). On summer squash, water soaking symptoms occurred on 85 % of
the tested fruit by 1 DPI. Watermelon, acorn squash, pumpkin, butternut squash, and
cucmnber were less susceptible with rare sporulation of P. capsici at 4 DPI (Figure 3-7E).
These fruit were more likely to develop water soaking symptoms that did not progress to
sporulation, even at 10 DPI (Figure 3-7B,C,F).

For almost all of the crops tested, susceptibility decreased with increasing fruit
size, but to varying degrees (Figure 3-8; P<0.01 for all crops except melon and
watermelon). Cucumber showed the most dramatic effect with near complete resistance

as the fruit developed (Figure 3-8A). Butternut squash, pumpkin, and acorn squash also

79

Figure 3-7. Response of different cucurbit fruit to inoculation with Phytophthora capsici.
A, D. Days after inoculation for 50 % of the ﬁ'uit to exhibit water soaked (A) or
sporulating lesions (D). B, C. Percent fruit showing water soaking symptoms at 4 (B)
and 10 (C) days post inoculation (DPI). E, F. Percent fruit showing sporulation at 4 (E)
and 10 (F) DPI. Each value is the mean of fruit from two or three replicate harvest dates
(where error bars are absent, the days to symptom development on 50 % of the fruits was
the same for each replicate date). Each bar is the mean i SE of 45-93 fruit. Bars with the
same letter are not signiﬁcantly different at P S 0.05 (LSD).

80

 

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Figure 3-8. Fruit size of ﬁeld-grown cucurbit ﬁ'uit vs. response to inoculation by
Phytophthora capsici at 4 days post-inoculation (DPI). Disease symptom scale: 1 - no
symptoms; 2 — water soaking; 3 - sporulation. Each point is the mean :1: SE of 2-8 fruit in
a given size class. Regression lines, r2 and P- values, and total number of fruits tested (n)
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82

 

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83

exhibited reduced susceptibility with increasing size, but not as completely as with
cucumber (i.e., fewer had no symptoms; Figure 3-8B-D). Watermelon, melon, zucchini,
and summer squash remained more susceptible. There was a modest decline for zucchini
and summer squash with increasing size, but the majority of fruit exhibited water soaking
(disease rating above 2; Figure 3-8E-H).

To more carefully examine the effect of fruit development on disease
susceptibility, greenhouse-grown, hand-pollinated butternut squash, pumpkin, and acorn
squash fruit of known ages were tested for response to P. capsici inoculation. All three
crops also showed strong age and size related decrease in susceptibility to P. capsici
(Figure 3-9), similar to what we had observed previously in the greenhouse for cucumber
(Gevens et al., 2006) and with ﬁeld fruit from this study. The majority of instances of P.
capsici sporulation occurred on fruit in the ﬁrst few days post-pollination, 0-3 DPP for
acorn squash, 0-5 DPP for butternut squash, and 0-8 DPP for pumpkin. Older fruit
exhibited water soaking without sporulation, or did not show any symptoms of infection.

The decrease in susceptibility corresponded with visible changes in exocarp
properties associated with fruit development (Table 3-1). Very young fruit of all crops
were green and very waxy, virtually all fruit at this stage produced sporulating lesions.
After a few days (3 -4 days for acorn squash and butternut squash, 10 days for pumpkin),
they lost wax but remained green. Susceptibility decreased between waxy green and
green fruit stages for all three crops where fruit developed water soaked lesions that did
not go on to sporulate. Each crop subsequently underwent color change/development
according to their maturity type. Size did not increase signiﬁcantly once color change

began (Table 3-1). Butternut squash had little decline after the green non-waxy stage; the

84

Figure 3-9. Relationship between fruit age and size and response to inoculation by
Phytophthora capsici at 4 days post-inocuation (DPI) for greenhouse-grown fruit. A, B,
C. Response of butternut squash (A), pumpkin (B), and acorn squash (C) fruit to P.
capsici inoculation with respect to fruit age and length. D, E, F. Mean disease rating (:1:
SE) of butternut squash (D), pumpkin (E), and acorn squash (F) fruit in relation to fruit
age. Each data point is the mean of 2-16 fruit. Disease rating scale: 1 — no symptom; 2
— water soaking; 3 — sporulation. Regression lines, r2 and P- values, and total number of
fruits tested (n) are indicated in each panel.

85

 

 

   

 

 

 

 

     

 

 

 

 

 

       

 

 

 

 

 

 

 

 

 

 

 

 

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87

majority of fruit still exhibited water soaking symptoms. For pumpkin, there was no
signiﬁcant difference in susceptibility between green and green/orange stage; however
there was further signiﬁcant decrease upon turning orange (water soaking decreased to 39
%). Acorn squash showed a further decrease in susceptibility when turning from green to
black as evidenced by a decrease in the percent ﬁ'uit exhibiting water soaking (59 % to 10
%).

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cucurbit fruits showed differences in susceptibility for zucchini, watermelon, butternut
and cucumber (Table 3-2). Signiﬁcant differences between peduncle and blossom ends
were also seen for greenhouse-grown butternut and acorn squashes. Greenhouse-grown
pumpkins, similar to the ﬁeld-grown fruit, did not show a signiﬁcant difference in

susceptibility between the peduncle and blossom ends.

88

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89

Discussion

Screening of approximately 150 cultigens did not identify a source of resistance
superior to the partial resistance already present within many commercial cultivars. The
screening process led to the observation that there are developmental differences in
cucumber ﬁ'uit susceptibility that can inﬂuence proper screening procedures and may also
inﬂuence effective disease management strategies in the ﬁeld.

Most ﬁ'uits younger than 10 DPP exhibited sporulation at 4 DPI, whereas fruit
older than 12 DPP generally remained symptom-free Interestingly, the transition from
susceptible to resistant appeared to coincide with the end of the period of rapid ﬁ'uit
elongation, suggesting that physiological changes associated with fruit growth and
development might explain changing susceptibility among cucumber fruit of different
ages. The effect of fruit age was observed for several different cucumber cultivars and
accessions. In addition, ﬁeld-grown cucumbers, harvested to test a range of fruit sizes,
exhibited the same trend as observed with greenhouse-grown fruits. These results
indicated that there are changes in the degree of susceptibility among fruits of different
ages in both greenhouse and ﬁeld conditions, and that the age-related decrease in
susceptibility is not genotype speciﬁc.

These observations have implications for appropriate screening procedures. If
harvested fruits are too mature, resistance may be observed which is not constitutive, but
is solely age-related. This matmity factor could be responsible for the failure to observe
resistance upon rescreening in several cases. The lack of signiﬁcant decline in

susceptibility between 7- and 14-day old fruit for two of the P13 may be due to genetic

9O

differences in susceptibility, or differences in rates of fruit development, such that 14
DPP ﬁ'uit are not physiologically equivalent among all PIs.

Age-related resistance (ARR) is becoming increasingly recognized as an
important component of plant defense against infection (Develey-Riviere and Galiana,
2007; Panter and Jones, 2002; Whallen, 2005). This phenomenon, which results in
increased resistance as young tissues develop, is distinct from the increase in
susceptibility that occurs in old, mature organs as the result of the ripening and
senescence. The mechanisms of AR are largely unknown. There is evidence that
salicylic acid is important to ARR in Arabidopsis response to Pseudomonas synringae
(Kus et al., 2002) and Hyaloperonospora parasitica (McDowel et al., 2005). AR in rice
(Oryza sativa) to Xanthomonas spp. (Century et al., 1999; Koch and Mew, 1991;
Mazzola et al., 1994) may be related to developmentally-regulated R gene-mediated
resistance which is controlled post-transcriptionally or by other mechanisms.
Developmentally-regulated expression of single-gene mediated resistance also has been
observed for potyvirus infection of cucumber seedlings (Ullah and Grumet, 2002; Wai
and Grumet, 1995). Pepper (Capsicum annuum) plants also showed ARR to infection by
P. capsici (Kim et al., 1989). The reduced susceptibility was suggested to be related to
physiological changes in root and stem tissues.

Similar to the response of cucumber fruit to P. capsisi, developing grape berries
(Vitis vinifera) also showed decreased susceptibility to infection by Unicinula necator,
causal agent of powdery mildew, and to Plasmopara viticola, causal agent of downy
mildew (F icke et al., 2002; Gadoury et al., 2003; Kennelly et al., 2005). Berries are

highly susceptible for the ﬁrst several weeks after anthesis. Developmental changes in

91

berries, such as brix content or morphological change of stomata to lenticels were thought
to be related to susceptibility changes, however, the mechanism has not been determined.

Examination of the age related resistance in cucumber fruit, showed that
resistance was associated with the outer 1-2 mm of fruit surface. Thin sections of
exocarp retained the resistance of susceptibility of the whole ﬁ'uit. More short germ-
tubes and appressoria, which are associated with rapid and successful penetration
(Grenville-Briggs et al., 2008), were formed on the 8 DPP fruit surfaces, whereas more
long germ tube and aberrant spores were found on the surface of 16 DPP fruits. This
difference could be due to cuticle properties as was observed to be a physical barrier to P.
capsici infection in New Mexican-type pepper (C. annuum) (Biles et al., 1993). Another
possibility is a difference in frequency of stomata, since Smith et al. (1979) reported that
stomata are almost absent from peduncle end compared to the blossom end, and are less
frequent in larger (older) ﬁ'uits relative to younger fruit. Whether the P. capsici
zoospores primarily enter through cucumber fruit stomata remains to be determined,
although that did not appear to be the case in the samples we obtained. Other
possibilities include changes in cell wall properties, production of defense compounds, or
induced resistance mechanisms. Further experiments will be required to determine the
properties responsible for the change in susceptibility that occurs during change as ﬁ'uit
develop. Consistent with resistance residing in the exocarp, seed development did not
inﬂuence the occurrence of AR in cucumber.

Other cucurbit fruit exhibited variability for overall susceptibility to P. capsici as
evidenced by number of fruits infected, time to sporulation, and extent of infection (water

soaking vs. sporulation). Summer squash, zucchini, and melon were the most

92

susceptible, whereas watermelon, acorn squash, pumpkin, butternut squash, and
cucumber were less susceptible. When comparing crops, susceptibility did not correlate
with taxonomic classiﬁcation. Of the four C. pepo crops, summer squash and zucchini
were highly susceptible, whereas acorn squash and pumpkin were less susceptible. Also,
thickness of rind did not correlate with the susceptibility, since muskmelon was one of
the most susceptible and cucumber one of the least.

Field and greenhouse grown cucurbit fruits tested in this study also showed a
general tendency of decreasing susceptibility with fruit development, but to varying
degrees. Acorn squash, butternut, and pumpkin exhibited reduced susceptibility with
increasing size, but not as complete as with cucumber. Despite the variations in fruit
types and sizes, the transition to reduced susceptibility, appeared to correlate with the end
of the rapid fruit elongation, as had been observed for cucumber fruit (Gevens et al.,
2006). Similar to the differences in overall susceptibility, zucchini and summer squash
remained highly susceptible even as they increased in size. The majority of the
watermelon and melon fruits also developed water soaking or sporulation symptoms
regardless of size.

Acorn squash, butternut squash, and pumpkin showed changes in exocarp features
that corresponded with reduced susceptibility. The transition from waxy green to green
exocarp was accompanied by a marked reduction in susceptibility, further suggesting that
changes in exocarp properties with fruit development might inﬂuence susceptibility,
although presumably many other changes are also occurring as this stage of development.

Another feature observed in cucumber, is a difference in susceptibility between

the peduncle and blossom end of the fruit. In Phytophthora infested ﬁelds, the blossom

93

end is more frequently infected than the peduncle end. While more frequent or earlier
contact with the soil may be a contributing factor to this observation, comparison tests of
both greenhouse and ﬁeld-grown fruit showed that the blossom end remained susceptible
longer than the peduncle end. These results indicate that there is a gradation of
susceptibility within the fruit such that the peduncle end of the ﬁ'uit becomes less
susceptible to P. capsici sooner than the blossom end. Cucumber fruit growth analyses
indicated that grth ceases at the peduncle end prior to the blossom end, suggesting that
differences in susceptibility may result from differences in physiological maturity
between two ends and corresponds with the age-related resistance observed in whole
fruit.

There also was a difference in susceptibility between peduncle and blossom ends
of fruit of several of the crops. The difference was most pronounced in zucchini and
butternut squash fruits. The results may reﬂect differences in the pattern of fruit growth
and maturation for the different species. Interestingly, the three (cucumber, zucchini, and
butternut squash) with the most signiﬁcant differences between fruit ends, have the most
elongated fruit shape, possibly allowing for a larger gradient in maturation between stern
and blossom ends.

In conclusion, cucurbit fruit exhibited an age-related decrease in susceptibility to
P. capsici where very young rapidly expanding fruit were the most highly susceptible.
There was a gradation of susceptibility within elongated fruit types such that the peduncle
end of the fruit became less susceptible to P. capsici sooner than the blossom end. In
cucumber this difference in susceptibility correlated with a difference in the period of

rapid elongation along the length of the fruit. Peel and zoospore germination analyses

94

indicate that the increased resistance in cucumber appears to be associated, at least in
part, with exocarp properties.

Information about age-related resistance can be helpful in deve10ping better
integrated pest management strategies (Develey-Riviere and Galiana, 2007; Ficke et al.,
2002). The above observations suggest the importance of protecting developing cucurbit
fruit ﬁom anthesis through the early period of rapid longitudinal growth. Timing of
chemical applications based when target tissue is most susceptible, could allow for more

effective disease control and also can minimize unnecessary chemical applications.

95

 

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means to reduce Phytophthora capsici disease incidence on cucumber fruit. J.
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Babadoost, M. 2004. Phytophthora blight: a serious threat to cucurbit industries. APSnet
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Biles, C. L., M. M. Wall, M. Waugh, and H. Palmer. 1993. Relationship of Phytophthora
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Bird, G., B. Bishop, E. Graﬁus, M. Hausbeck, L. Jess, W. Kirk and W. Pett. 2005. Insect,
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Century, K. S., R. A. Lagman, M. Adkisson, J. Morlan, R. Tobias, K. Schwartz, A.
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Develey-Riviere, M. P. and E. Galiana. 2007. Resistance to pathogens and host
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Phytologist. 175 :405-4 1 6.

F icke, A., D. M. Gadoury, and R. C. Seem. 2002. Ontogenic resistance and plant disease

management: A case study of grape powdery mildew. Phytophathology. 92:671-
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Gadoury, D. M., R. C. Seem, A. F icke, and W. F. Wilcox. 2003. Ontogenic resistance to
powdery mildew in grape berries. Phytopathology. 93:547—555.

Gevens, A. J ., K. Ando, K. Lamour, R. Grumet, and M. K. Hausbeck. 2006.
Development of a detached cucumber fruit assay to screen for resistance and

effect of fruit age on susceptibility to infection by Phytophthora capsici. Plant
Dis. 90:1276-1282.

Grenville-Briggs, L. J ., V. L. Anderson, J. Fugelstad, A. O. Avrova, J. Bouzenzana, A.
Williams, S. Wawra, S. C. Whisson, P.R. J. Birch, V. Bulone, and P. van West.
2008. Cellulose synthesis in Phytophthora infestans is required for normal
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Hausbeck, M. and K. Lamour. 2004. Phytophthora capsici on vegetable crops: research
progress and management challenges. Plant Dis. 88:1292-1302.

Iwaro, A. D., T. N. Sreenivasan, and P. Umaharan. 1997. Phytophthora resistance in
cacao (Theobroma cacao): Inﬂuence of pod morphological characteristics. Plant
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Kennelly, M. M., D. M. Gadoury, W. F. Wilcox, P. A. Magarey, and R. C. Seem. 2005.
Seasonal development of ontogenic resistance to downy mildew in grape berries
and rachises. Phytopathology. 95: 1445-1452.

Kim, Y. J ., B. K. Hwang, and K. W. Park. 1989. Expression of age-related resistance in
pepper plants infected with Phytophthora capsici. Plant Dis. 73:745-747.

Knerr, L. D., Staub, J. E., Holder, D. J ., and May, B. P. 1989. Genetic diversity in
Cucumis sativus L. assessed by variation at 18 allozyme coding loci. Theor. Appl.
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Koch, M. F. and T. W. Mew. 1991. Effect of plant age and leaf maturity on the
quantitative resistance of rice cultivars to Xanthomonas campestris pv. oryzae.
Plant Dis. 75:901-904.

Kus, J. V., Z. Zaton, R. Sarkar, and R. K. Cameron. 2002. Age-related resistance in
Arabidopsis is a developmentally regulated defense response to Psudomonas
syrinagae. Plant Cell. 14:479-490.

Lamour K. H. and M. K. Hausbeck. 2000. Mefenoxam insensitivity and the sexual stage
of Phytophthora capsici in Michigan cucurbit ﬁelds. Phytopathology. 902396-400.

Mazzola, M., J. E. Leach, R. Nelson, and F. F. White. 1994. Analysis of the interaction
between Xanthomonas oryzae pv. oryzae and the rice cultivars IR24 and IRBB21.
Phytopahtology. 84:392-397.

McDowell, J. M., S. G. Williams, N. T. Funderburg, T. Eulgem, and J. L. Dangl. 2005.
Genetic analysis of developmentally regulated resistance to downy mildew

(Hyaloperonospora parasitica) in Arabidopsis thaliana. Mol. Plant-Microbe
Interactions. 18:1226-1234.

Ngouajio, M., G. Wang, and M. K. Hausbeck. 2006. Changes in pickling cucumber yield
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Padley, Jr., L. D., E. A. Kabelka, and P. D. Roberts. 2009. Inheritance of resistance to
crown rot caused by Phytophthora capsici in Cucurbita. HortScience 44:211-213.

Padley, Jr., L. D., E. A. Kabelka, P. D. Roberts, and R. French. 2008. Evaluation of
C ucurbita pepo accessions for crown rot resistance to isolates of Phytophthora
capsici. HortScience. 43: 1996-1999.

Panter, S. N. and D. A. Jones. 2002. Age-related resistance to plant pathogens. Adv.
Botanical Res. 38:251-280.

Ristaino, J. B., G. Parra, and C. L. Campbell. 1997. Suppression of Phytophthora blight
in bell pepper by a no-till wheat cover crop. Phytopathology. 87:242-249.

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Smith, K. R., H. P. Fleming, C. G. van Dyke, and R. L. Lower. 1979. Scanning electron
microscopy of the surface of pickling cucumber fruit. J. Amer. Soc. Hort. Sci.
104:528-533.

Steel, R.G.D and J .H. Torrie. 1960. Principles and procedures of statistics. McGraw Hill
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Ullah, Z. and R. Grumet. 2002. Localization of zucchini yellow mosaic virus to the veinal
regions and role of viral coat protein in veinal chlorosis conditioned by the zym
potyvirus resistance locus in cucumber. Physiol. Mol. Plant Pathol. 60:79-89.

Wai, T. and R. Grumet. 1995. Inheritance of watermelon mosaic virus resistance in the
cucumber line TMG-l: tissue-speciﬁc expression and relationship to zucchini
yellow mosaic virus resistance. Theor. Appl. Genet. 91 :699-706.

Wang, G. and M. Ngouajio. 2008. Integration of cover crop, conservation tillage, and
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43:1770-1774.

Whallen, M. C. 2005. Host defence in a developmental context. Mol. Plant Pathol. 6:347-
360.

98

 

 

Chapter 4

Development of genomic analysis tools for early developing cucumber fruit

Abstract

Cucumber fruits are usually harvested and consumed at a young, immature stage.
Many studies have been published on fruit development of various species, however most
focus on late stages of development and important fruit quality attributes associated with
maturation and ripening, rather than early development. To increase understanding of
fruit development in cucumber, morphological and global gene expression analyses were
performed. Hand-pollinated greenhouse grown fruit at 0, 4, 8, 12, 16, 20, 26, and 32 days
post pollination (DPP) were monitored. Young cucumber ﬁ'uits (0-16 DPP) show
marked changes in fruit size, cell size, surface wax, chlorophyll content and patterns,
wart formation, spine development, and placenta and seed development. Pyrosequencing
technology ‘454’ analyses of rapidly growing 8 DPP fruit yielded 187,406 clean reads, of
which 88 % could be assembled into 13,879 contigs, 52-2,824 nt in length. The number
of sequences per contig, which is reﬂective of transcript abundance, ranged from 2-5,167.
BLAST analysis of the most highly represented transcripts against the nr protein
sequence NCBI database, indicated high representation of genes associated with protein
synthesis, ﬂowers, fruits or seeds of other species, latex related proteins, lipid
biosynthesis, cell expansion, defense, phloem transport, and photosynthesis. Results of

454 and qRT-PCR for selected genes sampled at different ages were comparable,

99

 

indicating that the 454 transcriptome sequencing can be used for analyzing relative gene

expression during ﬁ'uit development.

Introduction

Cucumber fruit rot caused by the soil borne Oomycete pathogen, Phytophthora
capsici results in severe yield loss that endangers the pickling cucumber industry
(Hausbeck and Lamour, 2004). In the preceding chapter, existence of age related
resistance (ARR) was revealed between cucumber (C ucumis sativus) fruit and P. capsici.
Inoculation of greenhouse grown fruits of known age showed that young, developing
cucumber fruit are highly susceptible to P. capcisi, while older fruit remained symptom
free. There was a sharp transition from susceptible to resistant that occurs in early fruit
development at around 10-12 days post pollination (DPP). The ARR appears to coincide
with the end of the rapid fruit elongation (Gevens et al., 2006), both among fruits of
different ages and along the length of individual fruits where the peduncle end stops
elongating sooner than the blossom end. This ARR was further observed from fruit
grown in the greenhouse and ﬁeld, and was also observed across several genotypes and a
variety of other cucurbit crops tested. Analyses of changes in gene expression during
fruit development may help us to gain insights into understanding the basis for this
phenomenon.

The development of next-generation sequencing technologies has dramatically
improved sequencing capability with respect to amount of data, time, and cost (Mardis,

2008; Margulies et al., 2005) and has allowed for new methods for gene expression

100

analysis. Roche (Branford, CT) introduced 454 pyrosequencing technology (Margulies et
al., 2005; Droege and Hill, 2008) which can sequence over 100 Mb in 7.5 hrs with
average reading of 200 bp. A new version of 454, GS F LX Titanium Series reagent, was
introduced in 2008 which can sequence over 400 Mb with average reading of 400 bp.

The length of reads generated by 454 pyrosequencing allow for conti g assemblies and
gene annotations of poorly characterized genomes. In addition, one of the applications of
the new sequencing technology is gene expression analysis, since the number of contig
reads represents frequency of the particular gene expression (Eveland et al., 2008; Ohtsu
et al., 2007; Torres et al., 2008). 454 sequencing also has capability of loading multiple
samples for a run, allowing side-by-side analysis. Gene expression analyses by 454
pyrosequencing results have been correlated with microarrays, northern blot analysis or
quantitative real time polymerase chain reaction (qRT-PCR) assays, suggesting high
reproducibility and suitability for gene expression analysis (Eveland etal., 2008). It also
can be used as an alternative to microarrays, especially for crops with small ESTs
numbers and/or no microarray platform available, such as cucumber.

Cucumber genomic tools are limited. The number of cucumber ESTs in the
National Center for Biotechnology Information (N CBI) database is only approximately
8,000 (4/15/2009 searched) compared to other species such as Arabidopsis - 1,728,728,
rice (Oryza sativa) - 1,260,123, and tomato (Solanum chopersicum) - 261,630. In the
past year, however, the cucumber genome was reported to be sequenced by a group at the
Chinese Academy of Agriculture Science and the Beijing Genomics Institute, Shenzhen
(Huang et al., 2009). It is anticipated that the 365 Mb sequenced genome will be publicly

available in the near future. Having sequenced genome and generating more ESTs will

101

 

be a tremendous advantage not only for contig assemblies and gene annotations, but also
for providing tools to study complicated biology of plants.

Model plants, such as Arabidopsis thaliana and rice (0. sativa) are well studied in
many aspects in plant development with the aid of tremendous information generated by
genomic tools. Fully sequenced genomes, microarrays, and large numbers of ESTs and
mutants are available. However, these species are not well suited to address questions
related to ﬂeshy fruit development. In case of ﬂeshy fruit, tomato (S. chopersicum), is a
well studied model plant (Carrari and Femie, 2006; Giovannoni, 2004; Moore et al.,
2002; White, 2002). The primary emphasis of fruit studies has been on late stage of
development and important fruit quality attributes associated with maturation and
ripening (Lee et al., 2007; Lemaire-Chamley et al., 2005). There has been less study
done on early ﬂeshy ﬁ'uit development, though this stage is essential for all fruit.
Deciphering early development of ﬂeshy fruit is important, since it inﬂuences yield and
quality of all fruits. Cucumber is one of the most important vegetable crops worldwide
that is harvested and consumed at a young, immature stage. Therefore, cucumber is
suitable to study early fruit development.

The objectives of the research presented in this chapter are to increase
understanding of early fruit development in cucumber by performing morphological and
global gene expression analysis. Morphological changes associated with cucumber fruit
development which could anchor the subsequent gene expression analyses were
catalogued for hand pollinated greenhouse grown fruits. Different cucumber fruit grth
stages (0, 4, 8, 12, 16, 20, 26, and 32 DPP) were collected to generate cDNA libraries for

454 pyrosequencing.

102

 

Materials and Methods
Cucumber fruit developmental morphological changes
Plant material and cucumber morphlogy

Three sets of ‘Vlaspik’ cucumber plants, planted on March 29, May 25, and June
14, 2007 were grown in the greenhouse. All sampling and data collection described
below were performed on each of the three experiments. Greenhouse conditions were as
described in chapter 3. To provide sufficient fruit of various ages developing under the
same conditions, for each experiment 80-184 ﬂowers from at least 80 plants were hand
pollinated on a single date. Only 1-2 fruit were set per plant to limit inter-fruit
competition effects on fruit development. The fruit were randomly assigned to groups of
10-20 for each harvest date. Prior to harvest, fruit were measured for length and diameter
and examined for external appearances including: presence or absence of wax along the
length of the fruit; wart development; color patterns (e. g., stripes); and changes in
presence, color, and densities of spines.

Nine to ten fruits ranked in the middle of the size range for each age group from
each experiment were harvested for further sampling for cDNA library construction.
Pericarp samples consisting of exocarp, mesocarp, and placenta tissue but no seeds, were
isolated from the middle of fruit by razor blade, immediately frozen by liquid nitrogen,
and kept at — 80 °C until RNA was isolated as described below. The cut ends of the fruit
were measured for pericarp and placenta diameter. Fruit sections adjacent to the cut ends
were dissected into approximately 1 cm3 pieces and ﬁxed in forrnalin-alcohol-acetic acid
(FAA) solution (Olmstead et al., 2007). Free-hand, thin sections (approximately 1 mm

thick) of transverse and cross sectional tissues of fruit from ﬁve fruit of each sample age

103

 

 

were isolated by razor blade. Each sample was measured for ﬁve neighboring
consecutive cells to obtain mean cell size and observed at three locations by light
microscope at 100x magniﬁcation with acridine orange dye (0.1 mg/ml). Impressions of
intact 8 and 16 DPP cucumber fruit epidermal tissue were prepared by the method
described in chapter 3.

Chlorophyll measurement

An additional ﬁve 1 g fruit excocarp samples (upper 1 to 2 mm) were removed by
conventional fruit peeler for chlorophyll measurement and stored at — 20 °C. Excocarp
samples were subsequently thawed at room temperature and blotted on paper to remove
excess water. One gram of isolated exocarp sections was immersed into N, N-
dimethylfonnamide for at least 24 hours at 4 °C in dark. Total chlorophyll was
calculated based on spectrophotometer absorbance measurements at 665 nm and 647 nm
(Inskeep and Bloom, 1985).

Library production and 454 sequencing.
Library construction 1.

The methods for RNA and cDNA sample preparation were adapted from the
procedures of Weber et al. (2007). For each age sample, RNA was pooled from 10 fruits.
Total RNA was isolated using the Qiagen RNeasy Plant Mini Kit (Qiagen, Valencia,
CA)or TRizol reagent (Invitrogen, Carlsbad, CA), treated with Qiagen RN ase free
DNAse, and assessed for quality by 2100 Bioanalyzer (Agilent, Santa Clara, CA) prior to
preparation of mRNA by Qiagen oligotex kit. mRN A was quantiﬁed by ﬂuorometer Q-
bit (Invitrogen) method and 30 ng of mRNA was used for cDNA construction. First

strand cDNA was synthesized using the Creator SMART cDNA library construction kit

104

(Clontech, Mountain View, CA) with modiﬁed CDSIII/3’ primer (Schilmiller et al.,
unpublished) to allow for improved sequencing efﬁciency. Titration PCR for the second
strand cDNA synthesis was performed for each sample to determine the best number of
PCR cycles as assessed by 1.1 % TAE agarose gel. Once the optimum cycle was
determined, second strand synthesis PCR was performed and quality assessed by 1.1 %
TAE agarose gel. In order to increase the quantity of cDNA, secondary PCR was
performed for 6-8 cycles. PCR products were puriﬁed by Wizard SV Gel and PCR
Clean-Up System (Promega, Madison, WI). Puriﬁed PCR products were digested with
SﬁI to remove primers and puriﬁed by Wizard SV Gel and PCR Clean-Up system. Final
concentration was assessed by the nanodrop ND-1000 method (Therrno Fisher Scientiﬁc
Inc., Wilmington, DE). Subsequent steps for 454 FLX pyrosequencing analysis was
performed by the RTSF Genomics Facility at Michigan State University (MSU). Each
sample (8 samples from 8 different DPP) was loaded on 1/ 16 454 Pico TiterPlate.
Library construction 2.

The same frozen 8 DPP pericarp tissues for the prior preparation were used for
library constructions. Total RNA was isolated by TRizol reagent, followed by DNase
treatment and clean up by RNeasy column. Quality of RNA was assessed by
formaldehyde gel (Sambrook and Russell, 2001) and nanodrop method. mRNA was
isolated as described earlier and was assessed with nanodrop method. 200 ng of mRNA
was used for cDNA construction. First and second strand cDNA synthesis was performed
as described above. Secondary PCR cycles were 10. For the 8 DPP — B sample, a
modiﬁed 5’ PCR primer which contains a SﬁI cut site was used

(AGTGGCCATTACGGCCGGG). The sample preparation procedure is illustrated in

105

Figure 4-1. Each sample (8 DPP-A and B) was loaded on a 1/4 plate. 454 F LX
pyrosequencing analysis was performed as described above.
Contig assembly and gene annotation.

Contigs of trimmed reads were assembled by the MSU bioinformatics group
using The Institute for Genomic Research (TIGR) clustering tools pipeline. Reads from
all samples were pooled to maximize available sequence data. Sequence data were
subjected to BLAST analysis in the green plant subdivision of the NCBI nr protein
database to search for homology to previously identiﬁed genes, to assist in contig
assembly, and to assign possible gene functions. To compare relative abundance between
samples, number of reads for a sample is divided by the total number of reads for the
sample and then multiplied by 1,000.

Transcriptome analysis

To analyze gene ontology (GO), highly abundant transcripts (20.15% transcript

frequency) were subjected to BLASTX analysis in The Arabidopsis Information
Resource (TAIR V7.0) database to search for homologies in Arabidopsis. Subsequently,
The Classiﬁcation SuperViewer Tool w/Bootstrap web database (Provart and Zhu, 2003)
was used for GO categorization.

Highly expressed transcripts (> 0.2 % frequency) also were subjected to BLAST
analysis against the International Cucumber Genomic Initiative (ICuGI) EST database to
search for presence or absence in current libraries from cucumber ﬂower buds, ﬂowers,
and fruit; melon fruit; and watermelon fruit. Presence or absence data were clustered and
visualized using Cluster 3.0 and TreeView 1.1.3 software (Eisen et al., 1998; de Hoon et

al., 2004; Saldanha, 2004).

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qRT-PCR 'i.
Total RNA was isolated from the same frozen samples described above and

assessed for quality and quantity as above. RT reactions were performed using the High

Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). Gene-speciﬁc

primers were designed using Primer Express software (Applied Biosystems) ABI Prism

7900HT Sequence Detection System was used for qRT-PCR analysis. Power SYBR

Green PCR Master Mix (Applied Biosystems) was used for PCR quantiﬁcation. Actin

from C. sativus (N CBI DQ641117) was used as an endogenous control for normalization.

PCR products from each gene were quantiﬁed with reference to corresponding standard

CLII'VCS.

Results

Morphological changes during cucumber fruit development

The progression and timing of growth and developmental processes were highly
reproducible across all three experiments. Fruit growth occurred rapidly after
fertilization with most rapid elongation occurring between 2 and12 DPP (Figure 4-2A).
Expansion of fruit diameter was observed for somewhat a longer time between 2 and 17
DPP. Microscopic analysis of cell size, regardless of their orientation, also showed rapid
cell size enlargement between 4 and 12 DPP (Figure 4-2B-D). Thicker cell walls were

observed from 16 DPP than 8 DPP epidermal cells (Figure 4-2E and F).

108

 

 

     

 

 

 

 

 

 

14 6 140
A B
12 ~ 5 " 120
A E
E 10 ‘ - 3 A100
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E —o— Frurt length 3 0 40
+ Fruit diameter ' 1 u.
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0 20 r
O 5 10 15 20 25 30 35 o 4 a 12 16 20 26 32
Fruit age (DPP) Fruit age (DPP)

8 DPP

 

Figure 4-2. Cucumber fruit cell size in relation to fruit age. A. Fruit length and diameter
measured daily up to 34 days post pollination (DPP). Each data point is mean of 4—10
ﬁ'uit :t SE. B. Fruit cell size was measured from 0, 4, 8, 12, 16, 20, 26, and 32 DPP fruit.
Each data point is mean 1 SE of 30 fruits. Mesocarp cells from cross section of 4 (C)
DPP and transverse section of 8 (D) DPP fruit were taken at 100x with acridine orange
stain. Bars = 50 um. Epidermal tissues of 8 (E) and 16 (F) DPP ﬁ'uit were taken at 100x.

109

Morphological changes associated with fruit grth were observed for both “F
external and internal properties. At 0 DPP, deep ridges along the length of fruit covered
the surface of the fruit. Spines were randomly scattered relative to the ridges. Spine
densities were the highest at 0 DPP and decreased rapidly at 4-8 DPP due to fruit
expansion, then remained essentially constant aﬁer 12 DPP (Figure 4-3A). Spine color
was translucent light green at 0 DPP, then started turning yellow around 8 DPP. Around
12 DPP, spines started to turn white; most were white by 16 DPP at which time many
abscised from the ﬁ'uit surface (Figure 4-3A). Warts, which are formed at the base of
spines, were diminutive at 0 DPP, however, they rapidly developed to become highly
prominent at 4 DPP (Figure 4-3A). With further fruit expansion, the warts ﬂattened out
and were completely absent by 12 DPP. At anthesis (0 DPP), fruit were covered with
thick dull-looking powdery wax (i.e., bloom) (Figure 4-3B). The bloom disappeared ﬁrst
from the peduncle end around 4 DPP, then the blossom end by 8 DPP. Some 8 DPP ﬁ'uit
had lost bloom from the middle part of the ﬁ'uit and by 12 DPP, the bloom disappeared
from ﬁ'uits completely. Stripes and specks on the surface of the fruit were absent in 0-4
DPP fruit, but started to emerge around 8 DPP (Figure 4-3B). Total chlorophyll content
was the highest at 0-4 DPP, and then declined continuously until approximately 20 DPP
(Figure 4-4).

There also were obvious changes in internal fruit morphology. Both placenta and
pericarp rapidly expanded from 4-16 DPP. The rate and amount of expansion was very
similar for both tissues (Figure 4-5A). Visible placenta changes included development of

gelatinous texture and hardened seed coats (ﬁgure 4-5B). The mesocarp turned from

110

Odpp 4dpp 8dpp 12dpp

 

Figure 4-3. Surface property changes in early cucumber fruit development. Spine and
wart development (A) and surface wax (bloom) coverage and stripe development (B) in
growing cucumber fruit at 0, 4, 8, and 12 DPP fruit. Arrows indicate spine and wart.

111

 

 

1.4
1.2 -
1.0 i
0.8 -
0.6 -
0.4 -

0.2 -

Total chlorophyll (mg*g'1)

0.0 -

 

 

 

I I I I I I I

0 5 10 15 20 25 30 35
Fruit age (DPP)

Figure 4—4. Total chlorophyll content in relation to fruit age. Each data point is mean of
at least 15 fruit peel/samples d: SE.

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113

green to whiter green which coincided with the development of a jelly-like placenta ‘i . - -

texture around 8-12 DPP. Seeds coats became visible around 8 DPP and turned from
white to tan with thicker hard coats developing between 12 and 16 DPP.
Cucumber fruit library construction by 454 sequencing
Library construction 1
cDNA libraries from fruit samples at eight different ages (0, 4, 8, 12, 16,

20, 26, and 32 DPP) were each loaded onto a 1/16 section of Pico TiterPlate resulting in a
total of 18,349 clean reads yielding approximately 4 million base pairs (bp) (Table 4-1A).
The mean and median read lengths were 222-239 bp which was equivalent to the 454
FLX system standards. Each sample yielded variable results. The 20 DPP sample had
76 % clean reads which contained 8,573 reads, whereas the 32 DPP yielded only 4 %
clean reads which contained 89 reads (Table 4-1B). The average percent clean reads
h6wever, was very low (33%), necessitating a revised sample preparation method. The
majority of poor reads were concatemers of 5’ primers used for the cDNA library
constructions.
Library construction 2

Several changes were made to improve sequencing including: additional steps to
obtain cleaner total RNA; higher amount of mRNA for cDNA synthesis; and re-designing
a 5’ primer to include a Sﬁl cutting site. The second 454 run generated excellent overall
sequence data with a total of 187,406 clean reads and 41,658,727 bp (Table 4-2) and
mean and median read lengths of 221 -235 bp. The two technical replications of 8 DPP
samples were run side-by-side on a 1/4 plate of 454, both samples gave a high percentage

of clean reads (94%), indicating very little concatemerization in this run. There was an

114

 

 

 

 

 

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Table 4-2. Summary of 454 sequencing results for 8 days post pollination (DPP)
cucumber fruit transcript samples

 

 

8DPP-A 8DPP—8*
Clean reads (1/4 plate) 75,497 111,909
% clean (clean/passed) 93.9 94.4
Mean read length (bp) 224 221
Median read length (bp) 235 234
Total bases 16,920,666 24,738,061

 

*8DPP-B library was constructed by using modiﬁed 5’ PCR primer for cDNA synthesis

116

 

excellent correlation in transcript frequency for contig groups between the two technical
replicates indicating reproducibility of results (Figure 4-6).
Transcriptome analysis by 454 sequencing

A total of 1,818 contigs ranging 162 -1724 bp in length (average 377 bp) were
assembled using 53 % of total clean reads from the ﬁrst 454 run (data not shown). The
number of the sequences per contig, which is reﬂective of transcript abundance, ranged
from 2 -450 with an average of 5 transcripts per contig. Aﬁer the second sequencing,
both 454 runs were combined to maximize the number of sequences available for contig
assembly. Of the 205,755 reads, 88.4 % could be assembled into a set of 13,878 contigs
(Table 4-3). The mean contig length was 416 bp, ranging 52- 2,824 bp. The number of
the reads per contig ranged from 2 to more than 5,000, with a mean of 13 reads per contig.

The 126 most highly expressed contigs (e. g. transcript frequency 2 0.15 %),
which represented 0.9 % of the 13,878 total contigs, accounted for 36.8 % of total reads
(Table 44). Approximately 10 % of the highly abundant transcripts did not have a match
in the TIGR database, suggesting that these genes could be unique to cucumber fruit
(Figure 4-7). Among the most highly expressed genes were latex-related proteins, and
proteins associated with lipids, growth, translation, defense, phloem transport, and
photosynthesis (Table 4-4).

The most highly expressed cucumber fruit gene, Csf-2, with more than 5,000
reads, has high homology with a major latex-like protein gene from Arabidopsis based on
NCBI BLAST search (Table 4-4). Other major-latex like protein genes were also in the

most frequently expressed group, including the fourth most expressed gene. In addition,

117

 

 

 

12

 

 

 

 

12

8d-B (TPT)

Figure 4-6. Pairwise scatter plot and regression analysis for 8 d-A and 8 d-B samples
expressed in transcripts per thousand.

118

Tr—Tl “\1‘, \nv.

IIHLJ R~

Table 4-3. Overall summary of contig assembly from combined 454 runs.

 

Total reads assembled into contigs 205,755
Number of contigs 13,878
Mean contig length (bp) 416
Range of contig length (bp) 52 - 2,824
Mean number of reads per contig 13

Range of number of reads per contigﬁ 2 - 5,167

119

 

I other biological processes

I other cellular components

[3 other metabolic processes

I other cellular processes

I other molecular functions
protein metabolism

I other membranes

I other intracellular components
I other cytoplasmic components
I] structural molecule activity

D other binding

El ribosome

transport

B cell organization and biogenesis
I response to stress

13 response to abiotic or biotic stimulus
I chloroplast

I other enzyme activity

I protein binding

r3 plastid

developmental processes

I cytosol

I nucleus

I transporter activity

I signal transduction

I mitochondria

I electron transport or energy pathways
I hydrolase activity

I DNA or RNA binding

I cell wall

I plasma membrane

I nucleic acid binding

I DNA or RNA metabolism

I:I transferase activity

I nucleotide binding

I kinase activity

I ER
E no hits found

 

Figure 4-7. Predicted gene ontrogy (GO) classiﬁcation from BLAST analysis of
transcript represented at frequency of > 0.1% against TAIR 7.0 database.

120

Table 4-4. Predicted function (based on BLAST analysis) and number of reads for highly

expressed cucumber ﬁ'uit genes (20.15% transcript frequency)

 

 

Hit Description - Predicted function E-value Overall Total
Reads
Csf-2 [Cucumis sativus] Latex-like protein 1.0E-87 5,167
24-sterol C-methyltransferase [Gossypium hirsutum] 1.E-165 1,992
lipid transfer protein [Prunus dulcis] 4.00E-16 1,790
MLP423 (MLP-LIKE PROTEIN 423) [Arabidopsis 2.00E-48 1,726
thaliana]
Peroxidase 1 .00E-169 1,716
aquaporin [Ricinus communis] 1.E-122 1,526
proﬁlin [Cucumis melo var. reticulatus] 2.0E-69 1,300
lipid transfer protein isoform 1 [Vitis vinifera] 3.0E-30 1,197
no hits found n/a 1,187
auxin-repressed protein-like protein ARPl [Manihot 1.0E-45 1,114
esculenta]
phloem ﬁlament protein; PPl; phloem protein 1 1.0E+00 1,079
[Cucurbita maxima]
putative histone H2B [Arabidopsis thaliana] 4.00E-16 1,059
bimodular protein [Medicago sativa] 1.0E+00 1,053
acidic ribosomal protein P3 [Corchorus olitorius] 9.00E-43 1,022
no hits found n/a 819
SPlLl (SPIRALl-LIKEI) [Arabidopsis thaliana] 8.00E-32 816
no hits found n/a 776
catalase [Cucurbita pepo] -2018.0 71 1
60S ribosomal protein L21, putative, expressed [Oryza 6.0E-86 702
sativa (japonica cultivar-group)]
ribosomal protein L30e [Pisum sativum] 3.0E-56 692
alpha-tubulin [Prunus dulcis] -1800.0 663
no hits found n/a 654
putative developmental protein [Nicotiana 2.00E-31 649
benthamiana]
putative chloroplast chlorophyll a/b-binding protein 1.E-150 642
[Carya cathayensis]
protein induced upon tuberization [Solanum demissum] 1.0E-24 637
type-2 metallothionein [Citrullus lanatus] 3.0E-40 585
S-adenosyl-L-methionine synthetase [Elaeagnus -1574.0 573
umbellata]
no hits found n/a 563
translation elongation factor 1A-2 [Gossypium 883 541
hirsutum]
vacuolar H+-ATPase c subunit [Citrus unshiu] 316 538
ribosomal protein L15 [Elaeis guineensis] 1.00E-109 518
Unknown protein [Arabidopsis thaliana] 1.0E-25 501
LTCORll [Lavatera thuringiaca] 8.0E-34 498
ADP ribosylation factor 002 [Daucus carota] 1.E-100 498

 

121

drill. H. mm «Eirt.ﬁrb lﬂu twain ululll sub AL «ll; 6

 

 

Table 4-4 (Continued from previous page.) E-value Overall Total
Hit Description - Predicted function Reads
no hits found n/a 487
proteasome maturation factor UMPl family protein 9.00E-53 451
[Arabidopsis thaliana]

DNA-binding protein, putative [Arabidopsis thaliana] 9.00E-53 439
thioredoxin h [Hevea brasiliensis] 2.0E-44 430
latex allergen 9.0E-11 418
lipase [Gossypium hirsutum] 1.00E-150 406
calmodulin [Prunus avium] 3.00E-79 392
chlorophyll a/b binding protein [Cicer arietinum] 1.00E-106 386
14-3-3 protein homolog [Maackia amurensis] 1.E+00 372
60S ribosomal protein L19 [Capsicum annuum] 1.00E-101 371
plastidic cysteine synthase 1 [Solanum tuberosum] 1.00E-101 370
608 acidic ribosomal protein [Prunus dulcis] 8.00E-46 368
tonoplast intrinsic protein bobTIP26-l [Brassica 1.E-106 366
oleracea var. botrytis]

aquaporin 1 [Gossypium hirsutum] 1.E-150 365
ribulose bisphosphate carboxylase/oxygenase precursor 1.E-101 361
peptide

unnamed protein product [Vitis vinifera] 5.0E-89 359
26 kDa phloem protein [Cucumis sativus] 1.E-129 351
actin depolymerizing factor-like protein [Arachis 3013-66 349
hypogaea]

putative copper chaperone [Citrus cv. Shiranuhi] 8.0E-29 345
integral membrane family protein [Arabidopsis 4.00E-36 343
thaliana]

major latex-like protein 1 [Plantago major] 1.00E-l8 341
snakin-l [Solanum tuberosum] 6.00E-18 338
translationally controlled tumor protein-related protein 5.0E-89 337
[Cucumis melo]

lipid transfer like protein [Vigna unguiculata] 6.0E-24 329
arginosuccinate synthase family [Arabidopsis thaliana] 0 324
no hits found n/a 319
40S ribosomal protein 823 [Elaeis guineensis] 4.00E-76 311
chlorophyll a/b-binding protein 1.E-149 305
60S ribosomal protein L32A [Cucumis melo] 2.0E-48 299
four F5 protein-related / 4F5 protein-related 2.00E-20 297
[Arabidopsis thaliana]

chloroplast photosystem II PsbR [Prosopis juliﬂora] 2.0E-63 296
unnamed protein product [V itis vinifera] 1.E-143 296
histone HIC [Nicotiana tabacum] 4.0E-6O 290
putative ribosomal protein S29 [Oryza sativa (japonica 1.28E-26 287
cultivar-group)]

cyclophilin [Cucumis sativus] 2.0E-96 286
unnamed protein product [Xitis vinifera] 5013-57 283

 

122

tUCU

{KKK

m:

if

many lipid transfer protein genes and many genes encoding ribosomal proteins were in
the most highly expressed group.

Transcripts were compared to entries in the ICuGI EST database including
cucumber, melon, and watermelon ESTs. The majority of the most highly expressed
cucumber fruit genes were also expressed in melon or watermelon fruit (Table 4-5).
Many genes were also found in current cucumber fruit and ﬂower bud EST libraries, but
less in cucumber leaf. The cases where expression was not observed in the current
cucumber EST library likely reﬂect the very small number of deposits of cucumber fruit
sequence data. In most cases the genes not present in the ICuGI database were somewhat
less frequently expressed in the 454 sequencing result, and so perhaps less likely to be
included in a small set of transcripts.

Verification of transcritome analysis by 454 sequencing by qRT-PCR

Selected highly expressed transcripts, which had 20 reads or more and differential
expression in rapidly growing young (4 DPP) fruit versus older, post-rapid expansion (20
DPP) fruit from the ﬁrst 454 run, were subjected to qRT-PCR analysis to verify transcript
frequency difference from fruit samples of different ages. Transcripts such as aquaporin
and chlorophyll a/b binding protein showed high expression (TTP) in younger fruit; while,
transcripts such as metallothionein, auxin-repressed protein-like protein (ARPl), and
catalase showed higher expression at later stage of development (Figure 4-8).

qRT-PCR results for the selected genes showed expression patterns similar to 454
sequencing results (Figure 4-8). Alpha tubulin, chlorophyll a/b binding protein, and

aquaporin genes were highly expressed in early fruit development (4 DPP).

123

 

’T-l

.AullI

Table 4-5. Presence/absence of the top 0.2 % cucumber fruit transcripts in the
International Cucurbit Genomic Initiative (ICuGI) EST libraries of cucumber fruit, leaf,

male and female ﬂower buds, melon fruit, and watermelon fruit.
cucumber lCuGl 454
Hit Description - Predicted function cuucmber ﬂower cucumber cucumber melon watermelon
leaf buds fruit fruit fruit frUIt
Csf-2 [Cucumis sativus] .‘ . + + . .
peroxidase ' . . + + . .

MLP423 (MLP—LIKE PROTEIN 423) [Arabidopsis
thaliana] _, + -
ribosomal protein L309 [Pisum eativum] ; +' ' -
no hits found . .
ribosomal protein L15 [Elaeis guineensis] 1 . +
bimodular protein [Medicago sativa] . +
acidic ribosomal protein P3 [Corchorus olItori us] ' . M +
+
+

I
4.

Unknown protein [ArabidopSIs thaliana] .

no hits foUnd . . .

no hits found ' - ' -
24-sterol C-methyltransferase [Gossypium hIrsutum] . .
auxin-repressed protein-like protein ARP1 [Manihot

++++++++++
+++++‘+i+++-+
++++++++.
++++++.

esculenta] . , . + + + +
putative histone H28 [ArabidopsIs thaliana] ‘ ' -1 i 7' - + + + +
latex allergen A 7 H . . + + + +
thioredoxin h [Hevea brasiliensis] V - l . + + + +
no hits found 7 7 I . 7 - + 17+ + 4»
protein Induced upon tuberization [Solarium . ‘ . + + + +
putative developmental protein [Nicotiana
benthamiana] . - + + + +
putative chloroplast chlorophyll alb-binding protein '

[Carya cathayensis] + + + + + -
608 ribosomal protein L21 putative expressed

[Oryza sativa (japonica cultivar—group” + . + + + .
chlorOphyll alb binding protein [Cicer arietinum] + l . + + + .

[aquaporin [Ricinus communis] + + + + . +

'alpha-tubulin [Prunus dulcis] + + + + + +
lipid transfer proteIn isoform 1 [Vitis vinifera] + +4 A + ' «I- + + '
tranSIation elongation factor 1A-2 [Gossypium ”
hirsutum] + + + + + +
type-2 metalIOthionein [Citrullus lanatus] + + + + + +
lipid transfer protein [Prunus dulcis] . + + + . +
profilin [Cucumis melo var reticulatus] . V i + -I- + . +
catalase [Cucurbita pepo] . + -I- + . +
no hits found . + . + . +

proteasome maturation factor UMP1 famIly protein
[Arabl do. . + . + . +
DNA~binding protein putative [Arabidopsis thaliana] ‘ - + . + - +

vacuolar H+-ATPase c subunit [Citrus unshiu] + . . + - +
calmodulin [Prunus avium] + . . + + +
SP1L1 (SPIRAL1- LIKE1) [Arabidopsis thaliana] - . - + + .
no hits found 7 -, 1 . + . + + .
Scadenosyi-Lﬁ-methio‘nine synthetaseIElaeag‘nus V 7' A
umbellata] - ‘ + . + + .
phloem filament protein; PP1; phloem protein 1
[Cucurbita maxi ma] . . . + + +
LTCOR11 [Lavatera thuringiaca] '7 i . I i . . 4- + +
ADP ribosylati on factor 002 [Daucus carota] . + - + + +
CBL-interacting protein kinase 9 [Populus V

’trichocarpa] . . . + - .
Iipase[Gossypium hirsutum] ‘ H ' W ‘ i ' l . , + . .

 

*+= EST was present in the library, - - EST was absent.

124

 

 

 

 

l

aquaponn
chlorophyll a/b

- 454
E2] qRT-PCR

alpha tubulin

l

vacuolar H+ ATPase

1

auxin repressed

catalase

J

metallothionein

 

 

 

 

lipid transfer .
26kDa phloem .
snakin ‘ W
40 -5 o 5 10 15 20

Fold-change I092 20dpp vs. 4dpp

Figure 4-8. Relative transcript levels of selected highly expressed genes in 20 vs. 4 DPP
fruit measured by 454 sequencing and quantitative PCR.

125

Auxin-repressed protein, catalase, and metallothionein showed somewhat greater
expression at 20 DPP vs. 4 DPP, while lipid transfer protein, 26 kDa phloem protein, and
snakin genes were much more highly expressed in later fruit development (20 DPP)
(Figure 4-8).

The selected genes were further examined by qRT-PCR for fruit at 4, 12, 20, and
32 DPP (Figure 4-9). Alpha tubulin, chlorophyll a/b binding protein, and aquaporin
showed high expression in younger fruit and decreased as fruit aged. Auxin-repressed
protein, catalase, metallothionein, and snakin showed low expression in young fruit and
increased as fruit aged. 26 kDa phloem protein, lipid transfer protein, and vacuolar H+

ATPase showed more variable patterns of expression.

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127

Discussion

Cucumber is a non-climacteric fruit consumed at an immature stage of
development while most fruits are consumed after maturation and ripening. For instance,
pickling cucumbers are typically harvested approximately 8 to 10 DPP when the fruit
attain desirable size for market, whereas tomato fruit are harvested usually at 35 DPP
(mature green stage) or later when the fruit start ripening (J anssen et al., 2008; Kader et
al., 1977; Miller and Wehner, 1989). Many studies of fruit development have been
directed toward later development and quality, however, there is increased interest to
study early fruit development since it sets a foundation for ﬁnal yield and quality (Lee, et
al., 2007; Mounet et al., 2009).

The early stages of cucumber fruit growth are marked not only by cell division
and rapid cell enlargement typical of fruit development in general (Gillapsy et al., 1993),
but also by other visible changes in morphology and development which highlight
dynamic developmental processes, especially during the period from anthesis (0 DPP) to
12 DPP (Figure 4-10). Early developing cucumber ﬁ'uit undergo loss of spines, warts,
and surface wax, chlorophyll degradation, and placenta and seed development. These
morphological and physiological changes provide context for gene expression analysis
during early fruit development. This time period also coincides with the stage at which
fruit are susceptible to infection by P. capsici (Ando et al., 2009; Gevens et al., 2006).
Fewer obvious external changes were observed later ages (after 12 DPP).

Challenges were encountered when constructing cucumber fruit cDNA libraries
for 454 sequencing. Poor recoveries of clean reads were obtained from the ﬁrst set of

454 samples. This has been commonly observed among cDNA libraries constructed for

128

 

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129

454 pyrosequencing by similar methods due to concatemerization of 5’ primers caused by
unknown mechanism(s) (Ali et al., 2009). Modiﬁcations were made to improve the
second 454 run. To obtain cleaner total RNA, additional steps were taken to remove
polysaccharides. Also, the quantity of total RNA was increased in order to increase
mRNA yield, and three-fold increased (30 ng to 200 ng) mRNA was used for cDNA
synthesis. To remove the region which can cause concatemers, a modiﬁed 5’ primer
which contains a SﬁI restriction site also was used. With these changes, the second 454
run yielded an excellent result.

Approximately 14,000 contigs were generated from cucumber fruit cDNA
libraries by 454 pyrosequencing. BLAST search in the NCBI database indicate that
many genes are also found in fruit of other species, and there are also genes that appear to
be unique to cucumber ﬁ'uit. The majority of the most highly expressed cucumber fruit
genes obtained from the 454 sequencing analysis were also present in other cucurbit
fruits. There was more overlap with cucumber ﬂowers than leaves as might be expected
since ﬁ'uits develop from ﬂoral tissues.

The most highly abundant transcript, Csf-2, also was found to be one of three
highly expressed genes in young cucumber ﬁ'uit samples at 3 days post pollination
(Suyama et al., 1999). Csf-2 was highly expressed throughout the cucumber ﬁ'uit
development as indicated by the ﬁrst 454 run. Its deduced sequence has high homology
to major latex-like proteins (MLP), and to a family of ﬂower and fruit speciﬁc genes.
MLP homologues were observed from fruit of other plants including opium poppy, bell
pepper, raspberry, strawberry, and tomato (Jones et al., 1998; Nam et al., 1999; Nessler et

al., 1994; Pouzeta-Romero et al., 1995; Ruperti et al.; 2002; Tsugane et al., 2005). MLP

130

was found to be localized to membrane bound vesicles in the laticifer of opium and to be i "T“
associated with small vacuoles close to the plasma membrane in bell pepper fruit
(Pouzeta-Romero et al., 1995; Grifﬁng and Nessler, 1989; Stromvik et al., 1999).
Cucumber, peach, and soybean MLP homologues were highly expressed in immature
fruit, while musk melon, strawberry, and raspberry homologues were expressed in
ripening ﬁ'uit, and tobacco and pepper homologues were expressed upon wounding
(Ruperti et al., 2002). The function of MLP is largely unknown, however there was a
linear correlation between peach MLP homologue (Pp-MLPI) mRNA accumulation and
fruit relative growth rate, suggesting that peach MLP is associated with fruit cell
expansion. Expression in fruits at various stages of development suggests possible
additional function of MLPs. MLP also has high homology to intracellular pathogenesis-
related protein (IPR or PR-lO) and major pollen allergen proteins, suggesting a role in
pathogen defense (Osmark et al., 1998; Stromvik et al., 1999).

Interestingly, Csf-2 was not seen in melon or watermelon fruit according to the
ICuGI database. It should be noted though that genes may not have been present in the
ICuGI database due to limited EST collection sizes, although if it were expressed at a
comparable level as in cucumber, we would expect it to be present in the melon and
watermelon databases. Other MLP genes were expressed in melon fruit (Aggelis et al.,
1997; Hadﬁeld et al., 2000), suggesting that related genes may play similar functions in
the different cucurbit fruits. Furthermore the majority of MLP homologues in melon,
including the Csf2 homolog, are expressed in root or phloem tissue (Aggelis et al., 1997;

Hadﬁeld et al., 2000; ICuGI database).

131

One emerging application of 454 sequencing studies is gene expression analysis
as an alternative to methods such as microarrays, northern blots and subtraction cDNA
libraries (Shendure, 2009; Vera et al., 2008). 454 pyrosequencing has many advantages
including reduced cost relative to developing microarrays for non-sequenced species,
recovery of rare transcripts, lack of necessity to clone into vectors, and massive number
of gene sequences which can be analyzed side-by-side from multiple samples (Shendure,
2008; Vera et al., 2008).

Analysis of a selected set of transcripts over the time period from 4-32 DPP fruits
by qRT-PCR showed that genes can be grouped by patterns of expression which may
provide information about the function and the importance of various gene products at
different stages of fruit development. For example, alpha tubulin, chlorophyll a/b
binding protein, and aquaporin genes are highly expressed in early fruit development (4
DPP) but not in older fruit. These three genes were frequently observed in early
developing ﬁ'uit of other species and all belong to gene families that appear to be
associated with rapid growth (Janssen et al., 2008; Lee et al., 2007; Lemaire-Chamley et
al., 2005; Schlosser et al., 2008; Wechter et al., 2008). Alpha tubulin which is related to
formation of cytoskeleton was highly expressed in developing watermelon and apple
(Janssen et al., 2008; Wechter et al., 2008). Chlorophyll a/b binding protein which has
function in photosynthesis was highly expressed in early developing apple (Lee et al.,
2007) as would be expected for young green fruit rather than mature fruit. Aquaporin,
which mediates water ﬂow across plant membranes during cell expansion, was highly
expressed in young grape berry and tomato fruit (Lemaire-Charnley et al., 2005;

Schlosser et al., 2008).

132

In contrast, auxin-repressed protein, catalase, metallothionein, and snakin genes
were more highly expressed in later fruit development (20 DPP). Auxin-repressed protein
was found in ripening apples and cassava root, however the function is unknown (Mir et
al., 2001; Reiley et al., 2007). Catalase is a detoxifying enzyme which scavenges
reactive oxygen species; it has been observed in apple and tomato fruit (Esaka et al.,
1997; Janssen et al., 2008). Metallothionein has been observed in many fruit at ripening
stage, including pineapple, banana, grape, and raspberry and is thought to have copper
tolerance and homeostasis function (Cobbett and Goldsbrough, 2002; Goes da Silva et al.,
2005; Jone et al., 1998; Liu et al., 2002; Moyle et al., 2005). Snakin, an antimicrobial
protein, was expressed in fully developed petals and carpels of potato ﬂowers as well as
tuber cork and medulla, and tomato exocarp (Lemarie-Chamley et al., 2005; Segura et al.,
1999)

Expression patterns of vacuolar H+ ATPase, lipid transfer protein, and 26 kDa
phloem protein were variable. Vacuolar ATPase expression was relatively constant
throughout the cucumber fruit development. Its function is thought to be involved in
cellular pH regulation and expression was induced upon ripening in tomato fruit (Moore
et al., 2005). Lipid transfer protein and 26 kDa phloem protein had a similar expression
pattern, high in 12 and 32 DPP and low in 4 and 20 DPP. Lipid transfer protein was also
expressed in fruits of pear, strawberry, grape, and tomato, and in most of the cases,
increased expression was observed in later development (Fonseca et al., 2004; Janssen, et
al., 2008; Tomassen etal., 2007; Yubero-Serrano et al., 2003). In tomato fruit, it was
isolated as one of the components of the polygaractonase (PG) multicomplex involved in

fruit cell softening, and was suggested to have a possible role as a modulator of the

133

activity and stability of cell wall based enzymes (Tomassen et al., 2007). The 26 kDa
phloem protein has a possible function in long distance translocation of protein (Gomez
et al., 2004), and was commonly observed in three genenra of cucurbits, Cucurbita,
Cucumis, and Citrullus (Read and Northcote, 1983).

The cucumber transcriptome data obtained from the 454 runs could be considered
as an 8 DPP pericarp transcriptome proﬁle (rapid growth stage), since the majority of
transcripts were sequenced from 8 DPP libraries from the second 454 rtm. In a tomato
transcriptional proﬁle from early fruit development (3, 6, 8, and 15 DPP), many of the
genes identiﬁed were absent from the previous tomato EST database including that early
fruit development genes were less well characterized (Lemaire-Chamley et al., 2005).
Genes with functions in fruit growth, such as those associated with accumulation of
soluble sugars and acids in vacuoles (e g., vacuolar invertase and phosphoenolypyruvate
carboxylase) and modifying cell walls (e. g., pectate lyase, pectinesterase, extensions, and
expansins) were highly expressed in the exocarp of developing tomato fruit. Genes with
functions in ﬁ'uit protection or protection from pathogens (e. g., lipid transfer protein,
chitinase and glucanase), also were highly expressed in the exocarp of young tomato fruit
which provides a barrier to the fruit. Genes involved in cell expansion by controlling the
ﬂux of water across the plasma membrane or vacuolar membrane (e. g., the plasma
membrane instrinsic protein, aquaporins), photosynthesis-related genes, and auxin- and
giberellin-controlled genes were highly observed in locules of early developing tomato
fruit. Many of these genes observed in young developing tomato fruit were also found in
the 8 DPP cucumber fruit trancriptome, suggesting fundamental similarities in early

ﬂeshy ﬁ'uit development.

134

In summary, theses results indicate that sequences obtained by 454
pyrosequencing will contribute to our understanding cucumber fruit genes in developing
ﬁ'uit. Comparative transcriptome analysis can be used to examine changes in gene
expression associated with different stages of development. Further analysis of different
ages and fruit tissues also may help to elucidate underlying mechanism in cucumber fruit-

P. capsici age related resistance.

135

 

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protein that responds to ABA, wounding and cold stress. J. Exp. Bot. 54:1865-
1877.

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Conclusions and future work

Cucumber fruit rot caused by the soil borne Oomycete pathogen, Phytophthora
capsici, is a serious problem in many parts of the US. Finding genetic resistance would
be the optimal method of disease control, however, no resistant cultivar or strong source
of resistance for breeding is available to date. Therefore, establishing alternative methods
to control the disease incidence, and understand P. capsici-cucumber fruit interaction
could provide insight into effective disease management.

In Chapter 1, I tested whether an alternate plant architecture could be helpful in
limiting P. capsici infection on cucumber. Based on my results, alternative cucumber
architecture resulted in changes in the canopy structure and temperature under the canopy.
However, the incidence of disease was signiﬁcantly higher than economically acceptable
level in many alternate architectures I tested. In other experiments, fruit from trellised
vines and the compact PI 308916, which holds young fruit off the ground had
signiﬁcantly lower disease incidence than other architecture types, suggesting that
reducing the contact of young cucumber fruit with soil can used as a tool to prevent P.
capsici infection. Thus, canopy conditions including temperatures under the canopy or
canopy structure are less important for disease control than preventing contact of the fruit
with the soil.

When screening fruit for resistance to infection by P. capsici, it was observed that
age/size appeared to greatly inﬂuence fruit susceptibility. To study this aspect more
carefully, greenhouse-grown, hand-pollinated cucumber fruit were inoculated with P.

capsici. Young and rapidly elongating cucumber fruit were most susceptible to P.

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capsici, but when they ceased elongation [approximately 10-12 days post pollination
(DPP)] they develop age-related resistance (ARR). AR was also observed in ﬁeld-
grown cucumber, other cucumber genotypes, other cucurbit fruit (acorn squash, butternut
squash, pumpkin, zucchini, watermelon, melon, yellow summer squash), and
parthenocarpic cucumber ﬁ'uit. The peduncle end of the ﬁ'uit became less susceptible to
P. capsici earlier than the blossom end, thus showing a gradation of susceptibility within
elongated fruit types. In cucumber, this difference in susceptibility correlated with
differences in the period of rapid elongation along the length of the fruit. Exocarp
properties at least partially appear to be responsible for the increased resistance in
cucumber, as was evidenced by zoospore germination analyses on exocarp section (peel).
Transcriptome samples from developing cucumber ﬁ'uit (0-32 DPP) were
analyzed by 454 pyrosequencing as a starting point to decipher underlying mechanisms
of age-related resistance in the P. capsici-cucumber-fruit interaction and to ﬁll the gap in
knowledge of early development of ﬂeshy ﬁ'uit. Additionally, these same ﬁ'uit used for
making cDNA libraries for 454 sequencing were used to catalogue morphological
changes in order to anchor changes in gene expression relative to developmental
processes. Dramatic morphological changes were observed both internally and externally
during early cucumber fruit growth. Developing cDNA libraries for 454 pyrosequencing
was challenging, however the modiﬁed method greatly improved quality of the result.
Approximately 14,000 cucumber fruit ESTs were obtained by 454 pyrosequencing.
Many of the genes highly expressed in developing cucmnber fruit were also observed in
the developing stages of other fruit such as tomato, apple, and grape. The cucumber fruit

transcriptomes obtained from chapter 4 and the recently sequenced cucumber genome,

143

 

can complement each other and can be used as a tool to gain insights in biology of
developing fruit, and to explore the basis of cucumber ﬁ'uit and P. capsici age related
resistance.

These ﬁndings provided both short- and long-term applications for cucumber
production. For immediate application, protecting ﬁ'uit from early development by
spraying fungicides can minimize disease incidence and may be applicable for many
cucurbit crops. Constructing cucumber fruit transcriptome libraries will be a tremendous
asset for not only understanding for immediate questions I presented here, but for future
questions.

In the future, more transcriptome data from other cucumber fruit development
stages (0, 4, 12, and 16 DPP) and other tissues including exocarp and mesocarp will be
needed for further analysis to understand the underlying age related resistance
mechanism and aspects in fruit development. Also, comparison between cucumber fruit
transcriptomes with other fruit ESTs will provide more insights into fruit development.

Cucumber ﬁ'uit transcriptomes from fruit highly susceptible (8 DPP) and resistant
(16 DPP) to infection by P. capsici can be compared. Transcripts that are uniquely
expressed in 16 DPP, including these that are absent or only present in 16 DPP, and
down- or up-regulated compared to 8 DPP will be candidate genes for further analysis.
In addition, since exocarp properties might be the sources for ARR in cucumber fruit to P.
capsici infection, the exocarp transcriptome of the peduncle end of 12 DPP fruit can be
compared to the exocarp transcriptome of the blossom end of 12 DPP ﬁ'uit. 12 DPP will
be of use since it showed intermediate response to infection by P. capsici, and also

showed difference in susceptibility between the peduncle and stem end. The mesocarp

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transcriptome from 12 DPP can be subtracted from the 12 DPP exocarp transcriptome to
gain information speciﬁc to exocarp. Literature available from oomycete- or
Phytophthora-plant interaction will be referred to in selecting possible candidate genes.

A complete set of cucumber transcriptome data from 0 to 16 DPP will be a useful
tool to study early fruit development since these time points cover the period from cell
division to cell enlargement which is a hallmark of early fruit development (Gillarpsy et
al., 1993). Comparison to each DPP transcriptome data as described above will be
implemented. Further, transcriptomes from other early developing ﬂeshy fruit such as
tomato, grape, and apple will be compared to cucumber transcriptomes. Morphological
markers which were already recorded in chapter 4 will also be anchored to gene

expression.

145

 

Literature Cited

Gillaspy, G., H. Ben-David, and W. Gruissem. 1993. Fruits: a developmental perspective.
Plant Cell. 5: 1439—145 1.

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