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EFFECTS OF ENVIRONMENTAL CONDITIONS ON
PHYTOPHTHORA CAPS/CI DISPERSAL AND DISEASE
DEVELOPMENT

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LEAH L. GRANKE

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EFFECTS OF ENVIRONMENTAL CONDITIONS ON PH YT OPHTHORA CAPSICI
DISPERSAL AND DISEASE DEVELOPMENT

By

Leah L. Granke

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Plant Pathology

2010

ABSTRACT

EFFECTS OF ENVIRONMENTAL CONDITIONS ON PH YT OPHTHORA CAPSICI
DISPERSAL AND DISEASE DEVELOPMENT

By
Leah L. Granke

The oomycete plant pathogen Phytophthora capsici Leonian can infect a variety
of hosts including vegetable crops in the Cucurbitaceae and Solanaceae. P. capsici has a
worldwide distribution and may be a signiﬁcant limiting factor to vegetable production
due to the root, crown, stem, and fruit rot and foliar blighting it causes. Laboratory and
ﬁeld studies were conducted to study the mechanisms of sporangia dissemination. Direct
laboratory observations showed .P. capsici sporangial dispersal occurred in water with
capillary force, but did not occur in response to wind or a reduction in relative humidity.
When airborne sporangial concentrations and environmental conditions were monitored
under ﬁeld conditions, sporangial concentrations were positively associated with rainfall.
Both laboratory and ﬁeld observations indicated that dispersal of sporangia via wind
currents is infrequent, and sporangia are unlikely to be naturally dispersed among ﬁelds
by wind alone.

Controlled laboratory studies were undertaken to determine the effects of water

temperature (2, 9, 12, 19, 22, and 32°C), spore concentration (1 x 102, 1 x 103, 5 x 103, 1

x 104, 2 x 104, and 4 x 104 zoospores/ml), and zoospore suspension age (0, l, 3, and 5

days old) on infection of pickling cucumbers by P. capsici. Zoospore motility and
mortality in response to commercial algaecides were also investigated. Cucumbers

became infected at all temperatures tested, except 2°C, and the highest infection

incidence was observed for cucumbers incubated in suspensions held at 219°C. More
fruits became infected when suspensions contained a greater number of zoospores.
While the incidence of fruit infection declined with the zoospore suspension age,
infection still occurred when 5-day-old suspensions were used. Commercial algaecides
inhibited zoospore motility and caused signiﬁcant zoospore mortality in laboratory assays
and show promise for treatment of infested irrigation water.

The effects of temperature (10, 15, 20, 25, 30, and 35°C) and relative humidity
(~35, 60, 70, 80, and 100%) on development of Phytophthora fruit rot of pickling
cucumber were investigated in controlled growth chamber studies. The effect of
wounding on disease development was characterized for small (2.0 to 2.5 x 8 to 9 cm),
medium (3.0 to 4.0 x 12.0 to 13.0 cm), and large (>4.5 cm x >14 cm) pickling
cucumbers. No lesions developed on cucumbers incubated at 10°C, but lesions were
observed on cucumbers incubated at all other temperatures tested. Disease severity was
greatest on cucumber fruits incubated at 25°C at 4 days post inoculation (dpi). Lesions
formed on cucumbers incubated at all relative humidities tested. The diameter of water-
soaking and pathogen growth increased as the relative humidity increased. Wounding
was found to lessen size-related resistance in pickling cucumber. The diameter of water-
soaking was similar for all wounded cucumbers at 4 dpi regardless of fruit size. While
sporangia were formed on wounded large fruits, greater sporangial production was
observed in lesions on small and medium fruits. These results indicate P. capsici is
capable of infecting cucumbers under a wide range of temperature and relative humidity

conditions.

To my parents, Gayle and Ray Granke,

for their limitless support and love.

ACKNOWLEDGEMENTS

I am very grateful to Dr. Mary Hausbeck, my major professor and mentor, for her
guidance, support, and for the many opportunities she has created for me to grow as a
scientist.

I extend sincere thanks to my co-workers in the Hausbeck Lab for technical
support, critical feedback, and for making work fun. My most sincere thanks to Lina
Quesada-Ocampo, my co-worker and dear friend, for critical feedback, inspiration, and
for helping me see the bigger picture. Special thanks also to Soﬁa Windstam for
guidance, to Shiela Linderman for manuscript review, to Blair Harlan for greenhouse
assistance, to Brian Cortright and Jen Foster for ﬁeld help, and to Bryan Webster for
keeping the lab organized. I would also like to thank the many undergraduates who
worked diligently with me to generate this research, including Kyle Heslip, Amy Lebeis,
Mitch Wood, and Ronnie Heslip. Additional thanks to Wei Wang for statistical advice.

Thank you to the members of my committee: Drs. Ray Hammerschmidt, Jay Hao,

and Mathieu Ngouajio for encouragement and critical analysis of my work.

TABLE OF CONTENTS

LIST OF TABLES ............................................ I ........................................................... vii
LIST OF FIGURES ...................................................................................................... viii
LITERATURE REVIEW ................................................................................................ 1
CHAPTER I: Dispersal and Movement Mechanisms of Phytophthora capsici Sporangia
Abstract ............................................................................................................. 17
Introduction ....................................................................................................... 1 8
Materials and methods ....................................................................................... 20
Results ............................................................................................................... 27
Discussion ...................................................... - ................................................... 34
Literature Cited .................................................................................................. 39

CHAPTER 11: Effects of Temperature, Concentration, Age, and Algaecides on
Phytophthora capsici Zoospore Infectivity

Abstract ............................................................................................................. 42
Introduction ....................................................................................................... 43
Materials and Methods ....................................................................................... 46
Results ............................................................................................................... 52
Discussion ......................................................................................................... 55
Literature Cited .................................................................................................. 64

CHAPTER 111: Effects of Temperature, Humidity, and Wounding on Development of
Phytophthora rot of cucumber fruit

Abstract ............................................................................................................. 69
Introduction ....................................................................................................... 70
Materials and Methods ....................................................................................... 71
Results ............................................................................................................... 78
Discussion ......................................................................................................... 89
Literature Cited .................................................................................................. 94

vi

LIST OF TABLES

Table 1.1. Number of direct observations of Phytophthora infestans or Phytophthora
capsici sporangia made with a dissecting or inverted compound microscope. Each cluster
of sporangia on an individual leaﬂet or cucumber was observed until dispersal occurred
or up to 2 min. ............................................................................................................... 28

Table 2.1. Algaecide treatments applied to water containing Phytophthora capsici isolate
SP98 zoospores and the resulting pH of the water .......................................................... 49

Table 3.1. Average, maximum, and minimum relative humidities observed in incubation
chambers over the course of this study. ......................................................................... 75

vii

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

Figure 1.1. A, Pickling cucumbers with actively sporulating Phytophthora
capsici were used for both laboratory and ﬁeld experiments. B, Close-up of a
sporangial lawn and C, sporangia stained with trypan blue on a cucumber fruit.
D-E, Volumetric spore sampler and platform supporting cucumbers with
sporulating P. capsici in a ﬁeld. F, Mini-chamber (on inverted compound
microscope stage) used for direct observations of the effect of wind on P. capsici
sporangia. .......................................................................................................... 23

Figure 1.2. A -B, Direct observation of individual Phytophthora infestans
sporangia being dispersed and C-E, settling on the bottom of the incubation unit.
A sporangium in the circled cluster of sporangia in A is dispersed and absent in
the subsequent video frame captured 0.06 5 later in B. A sporangium (arrow) in E
attaches to the bottom of the incubation unit after its shadow is seen in the
previous frame (D) ............................................................................................. 29

Figure 1.3. A Phytophthora capsici sporangium on a piece of cucumber fruit A,
before and B, after being exposed to 1.5 liters/min of wind for 60 s. No dispersal
was observed. Note dehydration of sporangium. ............................................... 30

Figure 1.4. A, A Phytophthora capsici sporangium before introducing water that
is distributed via capillary force along the surface of a cucumber fruit piece with
the sporulating pathogen. B, The sporangium detaches and is dispersed in the
water resulting in an empty sporangiophore. ...................................................... 31

Figure 1.5. Relationship between daily cumulative concentration of airborne P.
capsici sporangia (sporangia/m3/day) and the correspondent daily rainfall (mm)
for A, monitoring in the commercial cucurbit ﬁeld in 2006 and B, monitoring in

the experimental ﬁeld setting in 2008. Note the differences in scale on the y-axes.
.......................................................................................................................... 33

Figure 2.1. Effects of Phytophthora capsici zoospore inoculum concentration
and A, temperature (data from 2°C not shown as no infection was observed), and
B, zoospore age on infection of cucumbers. Each column represents the average
of three (A) or four (B) repeated tests with six replicate pickling cucumbers per
treatment per test. Error bars represent standard error. ...................................... 53

Figure 2.2. Effects of algaecide treatment and contact time on Phytophthora
capsici zoospore mortality (%). Each column represents the average of two
repeated tests with three replicate culture plates per test. Error bars represent
standard error. Columns with an asterisk are not signiﬁcantly different from the
positive control, bleach, at 230 min contact time (LSD, P>0.10). ....................... 56

viii

Figure 3.1. A, No symptoms of disease were observed on fruits incubated at any
temperature (25°C shown) at 1 dpi (Pl=point of inoculation). B, Water-soaking
(WS) was apparent on fruits incubated at 20 to 30°C at 2 dpi (30°C shown), but
pathogen growth outside of the point of inoculation (P1) was not observed.
Signiﬁcant pathogen growth and sporulation was apparent in water-soaked lesions
on fruits incubated at 20 to 30°C at 3 (C & D) and 4 dpi. ................................... 79

Figure 3.2. A, Mean water-soaked lesion diameter B, pathogen growth diameter
and C, number of sporangia produced on pickling cucumbers after 1, 2, 3, or 4
days incubation at 15, 20, 25, or 30°C following inoculation with Phytophthora
capsici zoospores. Note differences in y axes and break in the y axis from 10-100
in C. Error bars represent standard error. Bars that share letters are not
signiﬁcantly different within a time point (days post inoculation, dpi, lower case
letters) or between time points (capital letters). Response surface of the D, water-
soaked lesion diameter, E, pathogen growth diameter and F, number of sporangia
per lesion as a function of temperature and time (dpi). The plots were generated
using predicted values from ﬁtted loess nonparametric regression models.

Models were ﬁtted using 32 observations (two experiments x 16 treatment
combinations). ................................................................................................... 81

Figure 3.3. Mean A, water-soaked lesion diameter B, pathogen growth diameter
and C, number of sporangia produced on pickling cucumber fruits after 3 or 4
days incubation at 35, 60, 70, 80, or 100% relative humidity following inoculation
with Phytophthora capsici zoospores. D, Mean water-soaked lesion diameter,
pathogen growth diameter, and number of sporangia produced on pickling
cucumbers following 3 or 4 days incubation. Bars that share letters are not
signiﬁcantly different within a time point (days post inoculation, dpi, lower case
letters) or between time points (capital letters). E, The number of sporangia
produced on cucumbers incubated at 35, 60, 70, 80, or 100% relative humidity by
isolate. F, Mean water-soaked lesion diameter, pathogen growth diameter, and
number of sporangia produced on pickling cucumbers following incubation under
different conditions of relative humidity. Bars that share letters are not
signiﬁcantly different for a speciﬁc isolate (lower case letters) or between
conditions of relative humidity (capital letters). Error bars represent standard
error ................................................................................................................... 84

Figure 3.4. Mean A, water-soaked lesion diameter, B, pathogen growth diameter
and C, number of sporangia produced on pickling cucumber fruits of various sizes
(small, medium, or large) that were wounded or not 4 days post inoculation (dpi)
with a droplet of water containing Phytophthora capsici zoospores. Note
differences in the y-axes. Means that share letters are not signiﬁcantly different
for an isolate (lower case letters) or between treatments (size*wounding, capital
letters). Error bars represent standard error ........................................................ 87

LITERATURE REVIEW

Phytophthora capsici Leonian is an oomycete plant pathogen in the Kingdom
Stramenopila (11,23). Although Phytophthora spp. resemble fungi morphologically, they
are phylogenetically distinct from true fungi. According to their evolutionary history,
Phytophthora spp. are more closely related to the heterokont algae (2). Oomycetes have
traits that are absent in the true fungi including cell walls composed primarily of cellulose
(B-glucan), a predominantly diploid life cycle, and the need for an exogenous source of
sterols for sporulation (23).

P. capsici was ﬁrst identified as the causal agent of chile wilt in New Mexico in
1922 (48). Foliar blight and root, crown, and fruit rot of cucumber (Cucumis sativus L.),
zucchini (C ucurbita pepo L.), squash (C ucurbiia pepo L.), eggplant (Solanum melongena
L.), tomato (Solarium lycopersicon L.), pepper (Capiscum annuum L.), pumpkin
(Cucurbita moschata Duchesne ex Poir.), melon (C ucumis melo L.), and watermelon
(Citrullus lanatus (Thunb.) M‘atsum & Nakai), are elicited by P. capsici. Erwin and
Ribeiro (23) reported that at least 49 plant species have been infected by P. capsici
worldwide. Recently, snap bean (Phaseolus vulgaris L.) (29), lima bean (Phaseolus
lunatus L.) (20) and fraser ﬁr (Abiesfraseri (Pursh) Poir.) (57) have been added as new
hosts, and weeds may be susceptible (25). Additional hosts that were infected under
laboratory conditions included beet (Beta vulgaris L.), Swiss chard (Beta vulgaris L.),
turnip (Brassica rapa L.), spinach (Spinacia ()leracea L.), and velvet-leaf (A buiilon
theophrasti Medik) (70).

P. capsici overwinters and survives long-term in the soil as thick-walled oospores,

which are produced by sexual reproduction (35,41 ,43). Sexual reproduction is common

in Michigan (42) and other regions of the United States (55) and is the primary source of
genetic variation in nature and allows for generation of fungicide resistance (41,43).
Since P. capsici is heterothallic, both mating types are required to be present for oospore
production in the soil or on or within host tissue (39,72). The pathogen can survive as
oospores in the soil for years, making crop rotations relatively ineffective for managing
the disease (45). Oospores are capable of germinating, even in the absence of nutrients,
to give rise to mycelia and/or sporangia (3,23,34). Phytophthora oospore germination is
affected by length of dormancy period, nutrition, light, temperature, soil water matric
potential, chemical treatments, and treatment with enzymes (10,34,59).

P. capsici has coenocytic hyphae (23). When environmental conditions are
favorable, the hyphae differentiate into long caducous pedicles, which support mostly
papillate, but occasionally semipapillate, limoniform clustered sporangia. Proper water
potential, aeration, temperature, light, inorganic salts, exogenous sterols, and protein and
RNA synthesis are required for sporangia production (4,23,59). Sporangia are usually
limoniform, but may also be subspherical, ovoid, obovoid, ellipsoid, spherical, fusiforrn,
pyriform or distorted depending on light,'nutrients, and other environmental conditions.
Once mature, the caducous sporangia may be dispersed by wind-driven rain or in
irrigation water, but are not likely to be dispersed between ﬁelds via the wind
(30,42,6 1 ,66).

The asexual propagules (sporangia and zoospores) are responsible for rapid
polycyclic disease development when the environment is favorable for disease (32).
When a sporangium comes into contact with free water, it differentiates to produce 20 to

40 heterokont reniform, uninucleate swimming zoospores, which maintain motility in

water for 5 to 10 h (23). The motility period is affected by temperature and is shortened
by temperatures greater than those optimal for growth and lengthened by temperatures
below those optimal for growth. Motility is also affected by the concentration of
zoospores present, with lengthened motility periods for suspensions with higher
concentrations (23).

Zoospores are negatively geotaxic in water and they utilize chemo- and
electrotaxis toward the host to favor zoospore contact with the host (15,74). It has been
suggested that receptor molecules on the surface of P. capsici zoospores receive
environmental signals that direct preinfection behavior (7). After zoospore-plant contact
is achieved, zoospores encyst (lose ﬂagella and develop a cell wall) and form germ tubes
(3). Zoospores store adhesive material in small vesicles, which is released upon
encystment to facilitate a ﬁrm attachment to the plant surface and to anchor the spores in
a favorable position "for host penetration (65).

P. capsici may penetrate host surfaces directly or enter through natural plant
openings such as the stomata or lenticels. A non-pectolytic protein produced by the
hyphae is capable of macerating host tissue and may play an important role in penetration
of the host epidermis and invasion of susceptible tissue (77). Hyphae grow intercellularly
through plant tissues and develop haustoria within host cells to absorb nutrients (47).
Under speciﬁc light and temperature conditions, some isolates have been known to
produce chlamydospores in some hosts (73), but chlamydospores are not typically
produced by P. capsici isolates from pepper or cucurbit hosts (60).

Development of disease depends on the three components of the disease triangle

being present: a virulent pathogen, a susceptible host and a favorable environment (52).

Differences in virulence and pathogenicity exist between different isolates of P. capsici
on various hosts (24,40,57,69). The amount of inoculum present also may affect disease
severity and whether or not infection occurs (46,58).

Susceptibility of the host is inﬂuenced by the cultivar, age, organ affected, and by
whether or not the host is wounded. Many attempts have been made to ﬁnd cultivars that
exhibit resistance to P. capsici. Screenings of cucumber germplasm have yielded some
varieties that exhibit limited lesion development and sporulation, but not complete
resistance (27). Two pumpkin cultivars, Danmatmaetdol and Lil Ironsides (Harris Moran
Seed Co., Modesto, CA) show quantitative resistance (46). Tolerance has also been
identiﬁed in a few tomato cultivars with commercially acceptable horticultural traits
(8,36), but more work is needed to identify sources of full resistance. Several
commercial bell pepper cultivars possess tolerance to P. capsici, but growers are reluctant
to use these because of marketability problems due to poor fruit shape (24) and a
potential correlation with silvering (76), the separation of the fruit cuticle from the
epidermis. It is known that host resistance in pepper is controlled by two distinct genes,
but little information exists regarding inheritance of resistance to P. capsici in most host
plants (46,56).

Some plant organs are more susceptible than others. For example, while
cucumber plants show a relative tolerance to P. capsici, cucumber fruits are very
susceptible (32). Previous experiments have shown that age and/or size are important
factors for infection of and sporangial production on cucumber (27) and pepper fruits (5),

and on pepper plants (38). Generally, disease was more severe on younger/smaller fruits

and plants. Wounding has been shown to negate age-related resistance in pepper fruits
(5).

Climatic conditions influence development of diseases caused by Phytophthora
spp. (22). The minimum, optimal, and maximum growth temperatures for P. capsici
isolates in culture media range from 6 tol2°C, 24 to 33°C, and 32 to >35°C, respectively
(23,51,68,71). Schlub found that a greater percentage of detached pepper leaves were
infected following incubation at 23°C as compared to 15 or 31°C when sporangial
suspensions were removed from the leaves 4 hours post inoculation (66). Disease
incidence and lesion length were greatest when inoculated pepper fruits were incubated at
27°C (6). While temperature does affect disease, rainfall is the most important
environmental factor affecting sporangial dispersal (30) and disease progress and
intensity (9,66).

A number of factors including temperature (21), nutrition (78), light (31), aeration
(53), availability of sterols (33), and relative humidity (32,66) are known to affect P.
capsici sporangial production. Sporangial production is optimal at 24 to 27°C (21).
While a limited number of sporangia may be produced in the dark, sporangial production
is much greater under conditions of light. An exogenous sterol source is necessary for
sporangial production (33) and more sporangia are produced in an aerobic environment
(53). Previous work by Lamour and Hausbeck (32) suggested that sporangial production
on slicing cucumber fruits was greater at 60 and 80% relative humidity than at >90%.
Sporangial production following incubation at 25°C was greater for pepper stem pieces
that were incubated at 100 or 97.5% relative humidity than at 92.5 or 87% relative

humidity (66).

Recommended management strategies to control the diseases elicited by P.
capsici involve integrated approaches that focus on using cultural practices to manage
water, using cultivars with resistance to the disease, avoiding introduction of the
pathogen to new areas and ﬁelds, and chemical control via fungicides (63).

Cultural control practices such as water management, crop rotation, bedding and
plasticulture, and avoidance of low-lying or infested ﬁelds have limited Phytophthora
infection and dissemination in some situations (32). Since Phytophthora diseases are
worsened under conditions of high soil moisture and/or standing water (10), water
management strategies are key. Many studies have demonstrated the importance of water
in the development (10,66) and spread of disease caused by P. capsici (l 3). Water may
be managed within a ﬁeld by planting into well-drained ﬁelds, using raised beds, using
black plastic mulch, trellising cucurbit plants, irrigating sparingly and using trickle
irrigation (13,32,63). Mowed cover crops on bare soil or straw mulch between rows may
be utilized to reduce splash dispersal of soilbome inoculum (50,62). The trellising of
smaller cucurbits keeps fruits off the ground and out of standing water, reducing the
probability of infection via splash dispersal. The variety of cultural modiﬁcations
accessible to large-acreage cucumber and winter squash producers in Michigan are
limited by reliance on mechanical harvesters (32).

Since P. capsici may be found in surface water used for irrigation (28,64,75),
susceptible crops should not be irrigated with surface water to avoid pathogen
dissemination (28). Zoospores are able to survive for weeks in surface water at ~25°C
(64), and detection of P. capsici in surface water appears to be related to water

temperature (28). While most growers prefer to use surface water to irrigate vegetable

crops because well water is more expensive and less plentiful, water from deep wells is
preferable since surface water may be contaminated with P. capsici (28).

Crop rotation may be integrated with other methods to control Phytophthora
diseases, but is not successful because of the long-term survival of oospores even in the
absence of a host (45). In practice, even rotations for longer than ﬁve years to
nonsusceptible crops have not proven successful. A. minimum of a three to four year
rotation to nonsusceptible crops is recommended, but growers may be reluctant to do this
because of the relatively low value of ﬁeld crops (32,45) and because soybeans may be
vulnerable (29). Recent reports of hosts that were previously thought nonsusceptible
indicate that rotation strategies may become further limited (20,57).

Very few fungicides provide economically acceptable control of the diseases
caused by P. capsici. When ﬁeld conditions are ideal for disease development, none of
the available fungicides eliminate P. capsici-caused diseases completely (32). The
systemic phenylamide fungicide metalaxyl was introduced in 1977 and is widely used to
control oomycete diseases (12,16,81). The primary mode of action of metalaxyl
(Ridomil) is to inhibit RNA synthesis in sensitive isolates by reducing the incorporation
of uridine into RNA (18,19). Metalaxyl is a racemic mixture; mefenoxam is the more
potent enantiomer (81). Metalaxyl limits sporulation and mycelial growth inside the host
tissue, but has little effect on sporangium and zoospore germination (12,18,19).
Resistance to mefenoxam has been observed in some isolates of P. capsici in Michigan
and other states (44,54). A single incompletely dominant gene controls mefenoxam
sensitivity in P. capsici on cucurbit hosts, according to in vitro crosses. Isolates may be

grouped as fully sensitive, intermediately sensitive, and fully resistant based on their

sensitivity to mefenoxam (44). Once resistance is established, the future usefulness of
mefenoxam in a ﬁeld may be severely limited. A study in Michigan showed that
mefenoxam resistance did not decrease in an agriculturally signiﬁcant time period (2
years) after mefenoxam selection pressure was removed (43). Since no management
beneﬁt is expected from applying mefenoxam to a ﬁeld with a signiﬁcantly insensitive P.
capsici population, ineffective applications can increase economic losses to a grower
(44,54). Part of designing an effective fungicide strategy to control P. capsici should
include alternation between fungicides to delay the development of fungicide resistance
(32). Other fungicides such as dimethomorph (17,67), zoxamide in combination with
mancozeb (79,80), and cymoxanil in combination with famoxadone (37) have been
registered for and show efﬁcacy against P. capsici.

In addition to fungicide applications, fumigation has been employed to manage
reduced P. capsici inoculum in the soil. The use of methyl bromide was phased out in
2005 except for critical use exemptions, which have been granted to Michigan vegetable
growers for a number of years (1). Treatments containing methyl bromide have been
shown to reduce P. capsici propagules in the soil (26), resulting in some degree of
disease control in the ﬁeld (32). Various altematives to methyl bromide have been
proposed including other fumigants and biofumigants (14,49).

In summary, while signiﬁcant advances have been made to understand and
manage the diseases elicited by P. capsici, this pathogen continues to threaten production
in major vegetable growing regions of the United States and elsewhere. To effectively
manage this disease, future research needs to focus on determining new sources of host

resistance, new fungicide products and application methods, and the relationships

between P. capsici and environmental conditions. The research objective of this
dissertation was to determine the effects of environmental conditions on P. capsici

dispersal and disease development.

10.

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I6

CHAPTER I: DISPERSAL AND MOVEMENT MECHANISMS OF

PHYTOPHTHORA CAPSICI SPORANGIA

ABSTRACT
L. L. Granke, S. T. Windstam, H. C. Hoch, C. D. Smart, and M. K. Hausbeck. 2009.
Dispersal and movement mechanisms of Phytophthora capsici sporangia.
Phytopathology 99: 1258- 1264.

Understanding the mechanisms of Phytophthora capsici sporangial dissemination
is paramount to understanding epidemic initiation and development. Direct laboratory
observations showed P. capsici sporangial dispersal occurred in water with capillary
force, but did not occur in response to wind or a reduction in relative humidity.
Atmospheric sporangial concentrations were monitored under ﬁeld conditions using a
volumetric spore sampler in a commercial cucurbit ﬁeld and in an experimental setting
where copious sporangia were continuously available in close proximity to the spore trap.
Dispersal was infrequent (0.7% of total hours monitored) during sampling in a
commercial ﬁeld; 14 sporangia were detected during a 7.5-week sampling period. In the
experimental ﬁeld situation, dispersal occurred in 4.6% of the hours sampled, and 438
sporangia were impacted onto tapes during a 7-week sampling period. Airborne
sporangial concentrations were positively associated with rainfall at both sites, but not
vapor pressure deﬁcit. Furthermore, in the experimental ﬁeld situation, wind speed was
not signiﬁcant in regression analysis. Wind speed was not measured in the commercial
ﬁeld. Hence, both direct laboratory observations and volumetric spore sampling indicate

that dispersal of sporangia via wind currents is infrequent, and sporangia are unlikely to

be naturally dispersed among ﬁelds by wind alone.

17

INTRODUCTION

The oomycete plant pathogen Phytophthora capsici Leonian has a large host
range including vegetable crops in the Cucurbitaceae and Solanaceae (19) as well as snap
bean (Phaseolus vulgaris L.) (23), lima bean (Phasealus lunatus L.) (12), Fraser ﬁr
(A biesﬁaseri (Pursh) Poir.) (39) and weed hosts (22). P. capsici has a worldwide
distribution (19) and may be a signiﬁcant limiting factor to vegetable production (29).
Although P. capsici is a soilbome pathogen, it also causes signiﬁcant disease on aerial
plant tissues including rot ofthe fruit, stem, and crown and blighting of the foliage, in
addition to root rot (29,43). P. capsici may generate copious amounts of asexually
produced caducous sporangia on a host surface after infection has been established.
Mature sporangia may germinate directly via formation of one to several germ tubes or,
when in contact with free water, will differentiate into 20 to 40 biﬂagellate motile
zoospores (5). Abundant sporangial production is a key ecological trait that allows P.
capsici to exhibit rapid polycyclic disease development under favorable conditions (29).

Since spores are microscopic and hence difﬁcult to detect, especially at low
concentrations (4), most evidence for P. capsici dispersal has been indirect, either
through inferences based on spatial analysis of disease patterns (40,42) or genetic
diversity data (35,36). Studies in which the genetic diversity of P. capsici populations in
Michigan were examined suggested that long-distance dispersal of sporangia is
uncommon (35,36). Elucidation of the mechanisms of sporangial dissemination is
critical to fully understanding the progress of an epidemic within a ﬁeld. Several
dispersal mechanisms have been proposed for P. capsici, including transfer via (i) water

movement down rows, (ii) rain splash or wind-blown rain, (iii) air, or (iv) movement by

18

humans or invertebrate activity (29,42). Analysis of spatial patterns ofdisease support
water movement as a major dispersal mechanism for P. capsici propagules (40,42).
Splash dispersal has been observed for several Phytophthora spp. including P.
nicotianae. P. palmivora, and P. iaf‘estans (44,48). but has not been directly examined for
P. capsici even though it is assumed to be a means of pathogen spread. Although it has
been speculated that local aerial dispersal of P. capsici propagules may occur (43),
previous studies have described aerial dispersal as unimportant (41,46). To our
knowledge, the only study that has attempted to directly sample aerially dispersed P.
capsici propagules used media-containing Petri plates afﬁxed to stakes in a pepper ﬁeld
during an active P. capsici epidemic (46). This study found that aerial dispersal occurred
rarely, and dispersal in dry air was only detected when plates were within 5 cm of a
heavily sporulating source.

P. infestans, the causal agent of potato late blight is the most well studied
Phytophthora spp. with regards to aerial dispersal (11,32,34). It has long been known
that P. infestans sporangiophores display hygroscopic movement as relative humidity
decreases during the morning hours and actively release sporangia (14,32,33). Sporangia
are subsequently carried away from the inoculum source by wind (3). This mode of
dispersal is also seen in downy mildew pathogens such as Bremia Iactucae, the causal
agent of lettuce downy mildew (45). Because P. inf‘estans and P. capsici both infect and
form sporangia on above-ground plant tissue, growers sometimes postulate that P. capsici
sporangia may be dispersed in a manner similar to that of P. iafesrans. To our
knowledge, no studies have directly examined P. capsici sporangial detachment and

dispersal in response wind and water. The objectives of our study were to: directly

observe whether a change in relative humidity, direct wind, or water combined with
capillary force enables detachment and transport of P. capsici sporangia, and assess the

probability of aerial dispersal in commercial and experimental ﬁeld situations.

MATERIALS AND METHODS
Growth and maintenance of Phytophthora isolates. P. capsici isolates SP98
and 0664-1 were maintained at room temperature (25 d: 2°C) under continuous

ﬂuorescent lighting on UCV8 (160 ml unclariﬁed V8 juice, 840 ml distilled water, 30

mM CaCO3, and 1.5% agar) and were transferred to fresh UCV8 weekly. SP98 (A2

mating type, sensitive to mefenoxam) was isolated from pumpkin (Cucurbita pepo L.) in
Michigan, and 0664-1 (A1 mating type, sensitive to mefenoxam) was isolated from
pepper (Capsicum annuum L.) in New York. Isolate notation refers to the culture
collections maintained in the laboratories ofM. K. Hausbeck at Michigan State
University and C. D. Smart at Cornell University. P. infestans clonal lineage US-l 1 was
obtained from the laboratory of W. E. Fry at Cornell University and was maintained on
detached tomato leaﬂets (Solarium lycopersicum L. cv. Sunchief).

Inoculation. Detached pickling cucumber fruits (Cucumis sativus L., 10 i 1 cm
long) were surface disinfested with a 10% bleach (0.62% NaClO) solution for 10 min,
rinsed with distilled water, blotted dry and placed in a sterile humid chamber (23 x 10 x
31 cm lidded clear plastic box with moist paper towels in the bottom to maintain
humidity) before inoculation with P. capsici zoospores. Zoospore suspensions were
prepared by ﬂooding actively sporulating (4-7 day old) P. capsici cultures with sterile

distilled water and incubating the plates at 4°C for 45 min followed by 30 min at room

20

temperature. The concentration of zoospores was determined using a hemacytometer and

adjusted to 2.5 x 106 zoospores/ml for each isolate. Three IO-ul droplets of zoospore

suspension were used to inoculate each cucumber fruit. Cucumbers were incubated at
room temperature under continuous ﬂuorescent lighting until copious sporangia were
detectable by light microscopy, approximately 72 to 96 h post inoculation (Figure 1.1A-
C).

Tomato leaflets were inoculated by ﬁrmly pressing healthy leaﬂets against ones
with actively sporulating lesions caused by P. infestans. Inoculated leaﬂets were
incubated at 19°C in 100 x 15-mm round plastic Petri dishes with either water agar or
moist ﬁlter paper in the lids to maintain humid conditions (incubation units) and were
used once abundant sporulation was apparent, which was a minimum of 4 days post
inoculation.

Fabrication of mini-chambers for direct observation of P. capsici sporangia.
For preparation of mini-chambers, polydimethylsiloxane (PDMS, Sylgard 184, Dow
Corning, Midland, MI) was poured over a 100 x 100 x 15mm plastic Petri dish and
cured at 60°C for 1 h. Spacers were used to maintain a PDMS thickness of 5 mm.
Following curing, a square of PDMS was cut into an H-shape and released from the Petri
dish. To create the bottom half of the chamber, the PDMS and a 51 x 76-mm microscope
slide were covalently bonded to each other following exposure to an air plasma (15).
Openings were bored through the PDMS, and stainless steel tubing (Small Parts Inc.,
Miramar, FL) was inserted and sealed with unpolymerized PDMS, which was heat-cured
and reinforced with Torr Seal epoxy (Varian Inc., Lexington, MA) to create an inlet and

outlet for wind at each end of the chamber. A cover glass was placed on the mini-

21

chamber and held in place with microscope stage clips to seal the chamber during
observation. Wind was generated using a TopFin XP-125 aquarium pump (PetSmart,
Phoenix, AZ) and humidiﬁed by passing air through moist cheesecloth loosely packed
into a humidity chamber to minimize sporangia dehydration. The air ﬂow was adjusted
to 1.50 i 0.06 liters/min using a ﬂow meter (Dwyer Instruments, Michigan City, IN).
Vinyl and rubber tubing was used to route the wind from the pump through the humidity
chamber and into the mini-chamber.

Direct observation of sporangia exposed to wind and water. Sporangia were
directly observed using an Olympus SZX12 dissecting microscope (Olympus America
Inc., Center Valley, PA) with an Intralux 4000 ﬁber optic light source (Volpi,
Switzerland) for overhead illumination or an Olympus [MT-2 inverted compound
microscope. Images and real-time movies were captured using a CoolSnap Cf camera
(Photometrics, Tucson, AZ). MetaMorph software (Universal Imaging, Dowingtown,
PA) was used for shutter control, image capture, and subsequent image processing.
Individual video frames were obtained from a streaming video sequence.

Tomato leaﬂets with actively sporulating P. infestans were used as a positive
control to demonstrate that sporangial dispersal in the laboratory setting could be directly
observed. The top of the incubation unit was removed, which allowed low humidity
ambient air to enter the unit resulting in a humidity decrease in the unit. Sporangia were
directly observed and images were captured as described above. Intact pickling
cucumber fruits with P. capsici sporangia were removed individually from a moist

incubation chamber and placed onto a 103 x 83-mm glass cover slide to observe P.

22

 

 

Figure 1.1. A, Pickling cucumbers with actively sporulating Phytophthora capsici were

used for both laboratory and ﬁeld experiments. B, Close-up of a sporangial lawn and C,
sporangia stained with trypan blue on a cucumber fruit. D-E, Volumetric spore sampler
and platform supporting cucumbers with sporulating P. capsici in a ﬁeld. F, Mini-
chamber (on inverted compound microscope stage) used for direct observations of the

effect of wind on P. capsici sporangia.

23

 

capsici sporangia after a reduction in relative humidity. The fruit were placed on the
glass cover slide that was scanned before and after sporangial observation to ensure all
dispersal events were detected. Sporangia were observed for 2 min with images captured
as described above. A small piece of tissue containing sporangia was excised from the
fruit and placed into a mini-chamber. Sporangia were observed using a compound
microscope for 2 min while 1.5 liters/min of air was pumped through the mini-chamber

(Figure 1.1F).

 

The glass bottom of the mini-chamber was scanned before and after observation t
of sporangia to determine if any undetected dispersal occurred. Finally, two small pieces
oftissue containing sporangia were excised from the same fruit and placed onto a glass
microscope slide 1 to 2-mm apart with the infected surfaces facing each other. A cover
slip was placed on top of the tissue pieces and 10 to 50-u1 of water was pipetted between
the pieces allowing capillary action to draw the water down the length of the pieces. The
sporangia were directly observed for dispersal in water as described above. At least 30
observations were made for each potential dispersal event.

Monitoring of atmospheric sporangial concentrations of P. capsici under ﬁeld
conditions. Atmospheric concentrations of P. capsici sporangia were determined using a
Burkard 7-day volumetric spore sampler (Burkard Manufacturing C 0 Ltd,
Rickmansworth, Hertfordshire, UK). The sampling airﬂow rate was 10 liters/min, and the
sampler was set to allow free movement according to wind direction so that the oriﬁce
faced the prevailing wind. Sporangia were impacted onto tapes coated with an adhesive

mixture of petroleum jelly and parafﬁn (9:1, wt/wt) dissolved in sufﬁcient toluene to

24

result in a thick, liquid consistency. Tapes were removed weekly, cut into 48-mm
lengths, scored at hourly intervals, stained with aniline blue in glycerol (0.14 mg aniline
blue, 20 ml distilled water, 15 ml glycerol, 10 ml of 85% lactic acid), mounted on glass
slides under 22 x 50-mm cover slips, and sealed using Cytoseal (Richard-Allan
Scientiﬁc, Kalamazoo, MI). Slides were scanned, and P. capsici sporangia were
examined at 400x magniﬁcation and identiﬁed based on morphological characteristics
(50). ‘
Atmospheric sporangial concentrations were monitored in a commercial cucurbit

ﬁeld and in an experimental ﬁeld situation. Spore sampling was conducted for a period

 

of 7.5 weeks (16 June to 8 August 2006) at a commercial farm in Cass County, MI
planted to cucurbits (acorn squash (Cucurbita pepo L. cv. Table Ace) followed by
cucumber (cv. Cobra)). Fungicides were applied to acorn squash in support of fungicide
research that was conducted in this ﬁeld and to the cucumber crop according to
commercial recommendations. Severe disease was observed in the acorn squash, and
more than 75% of plants were dead by 26 July when the spore trap was moved to
cucumber.

Atmospheric concentrations of sporangia were monitored for 7 weeks (19 July to
16 September 2008) in an experimental ﬁeld setting at the MSU Muck Soils Research
Farm, Laingsburg, MI. At this site, infected cucumbers with sporulating P. capsici
(isolate SP98) were prepared as described above. Cucumbers were placed on a raised
platform approximately 15 to 40 cm from the sampler so that infected tissue with the
sporulating pathogen faced the Burkard spore sampler and was level with the sampler

oriﬁce (Figure 1.1D and E). This conﬁguration allowed for quantiﬁcation of dispersal

25

without the limitation of a canopy and with minimal spatial dilution effects. Fruit were
replaced with new infected cucumbers with sporulating P. capsici triweekly to ensure
that sporangia were constantly available. Cucumbers were checked for sporulating P.
capsici using a dissecting microscope before and after placement in the ﬁeld to ensure
that copious amounts of sporangia were available for the entire monitoring period.
Collection of weather data and statistical analysis. Hourly meteorological data
of temperature (°C), relative humidity (%), and rainfall (mm) were measured by a
Watchdog model 450 data logger, and tipping bucket rain gauge (Spectrum
Technologies, Plainﬁeld, IL) at the commercial ﬁeld. Hourly meteorological data of air
temperature (°C), relative humidity (%), average and maximum wind speed (m/s) and
rainfall (mm) were measured by an automatic weather station belonging to the Michigan
Automated Weather Network at the experimental ﬁeld. Vapor pressure deﬁcit (VPD,
kPa) was calculated for both sites using temperature and relative humidity values (38).
Statistical analyses were performed using the SAS statistical package version 9.1
(SAS Institute, Inc., Cary, NC). The PROC CORR procedure was used to determine any
positive or negative correlations among daily cumulative atmospheric sporangial
concentration, daily cumulative rainfall, and daily average VPD for the commercial ﬁeld
and to determine correlations between daily cumulative atmospheric sporangial
concentration, daily cumulative rainfall, daily average VPD, and daily average wind
speed prior to regression analysis for the experimental ﬁeld situation. Autocorrelation in
the data set was characterized using ITSM 2000 software and was detected for the
experimental ﬁeld situation (attached to (8)). Regression analysis of data from the

commercial ﬁeld was not completed as the assumptions of regression could not be met.

26

 

The PROC AUTOREG procedure of SAS was used for regression analysis of data from
the experimental ﬁeld to appropriately address the autocorrelation detected in this data
set. The AUTOREG procedure calculates the equivalent of the least square parameter
estimates of a regression model when the data are time series and the error term is an
autoregressive process. The AUTOREG procedure uses the two-step full transform
estimation method (28) to correct for the lack of independence through time of the error
term of time series data. Variables with P3 0.05 were considered statistically signiﬁcant.
The ﬁnal day of sampling at each site was not used in statistical analyses because less

than 24 h of data were available for these days.

RESULTS

Direct observations on wind and water effects on dispersal. P. infestans
sporangiophores displayed hygroscopic movement almost immediately after a reduction
in relative humidity. In all cases where mature sporangia were present, dehiscence
followed the twisting of sporangiophores under nearly still conditions (Table 1.1). All
dispersal events took place within 60 s of a reduction in humidity, and could be clearly
viewed by microscopy (Figure 1.2). This validated that the methods used in this study
were sufﬁcient to directly observe sporangial dispersal. Dispersal of individual sporangia
was rapid, often no longer than a single video frame (Figure 1.2 A-B). In contrast, P.
capsici sporangiophores did not display hygroscopic movement upon a reduction in
relative humidity, and dispersal was not observed (Table 1.1). Sporangia on the
cucumber fruit surface were not dispersed in wind (Table 1.1, Figure 1.3). However,
water in combination with capillary force caused sporangia to readily detach from the

fruit surface almost immediately upon wetting the cucumber fruit surface, and sporangia

27

Table 1.1. Number of direct observations of Phytophthora infestans or Phytophthora

capsici sporangia made with a dissecting or inverted compound microscope. Each cluster

of sporangia on an individual leaﬂet or cucumber was observed until dispersal occurred

or up to 2 min.

 

Observations (% with dispersal)

 

X

 

Pathogen Lineage/isolate Host tissue RH Wind Water
P. infestans us-11 Tomato leaﬂets 40 (100) n/ay m

P. capsici SP98 Cucumber fruit 30 (0) 30 (0) 30 (100)

P. capsici 0664-1 Cucumber fruit 30 (0) 30 (0) 30 (100)

 

x RH=a reduction in relative humidity

y n/a=not applicable

28

 

Figure 1.2. A -B, Direct observation of individual Phytophthora infestans sporangia
being dispersed and C-E, settling on the bottom of the incubation unit. A sporangium in
the circled cluster of sporangia in A is dispersed and absent in the subsequent video
frame captured 0.06 5 later in B. A sporangium (arrow) in E attaches to the bottom of the

incubation unit after its shadow is seen in the previous frame (D).

29

 

Figure 1.3. A Phytophthora capsici sporangium on a piece of cucumber fruit A, before
and B, after being exposed to 1.5 liters/min of wind for 60 s. No dispersal was observed.

Note dehydration of sporangium.

30

 

Figure 1.4. A, A Phytophthora capsici sporangium before introducing water that is
distributed via capillary force along the surface of a cucumber fruit piece with the
sporulating pathogen. B, The sporangium detaches and is dispersed in the water resulting

in an empty sporangiophore.

31

were transported in the water (Table 1.1, Figure 1.4). Videos capturing these dispersal
experiments may be viewed at the home page of M. K. Hausbeck.

Atmospheric P. capsici sporangial concentrations under ﬁeld conditions.
When atmospheric sporangial concentrations were monitored in a commercial cucurbit

ﬁeld, dispersal occurred during 0.8% of hours sampled (10 of 1278 h). The concentration

of sporangia never exceeded 5 sporangia/mJ/h when dispersal was detected. In total, 14

sporangia were impacted onto tapes during the course of the 7-week sampling period.
Only one sporangium was sampled during most (80%) of the hours when dispersal was
detected. In the experimental ﬁeld situation (Figure 1.1D), aerial dispersal occurred in

4.6% of the hours sampled (54 of 1 174 h). The concentration of sporangia was low (515
sporangia/mJ/h) for 80% ofthe occasions when dispersal was detected. Only 438

sporangia were impacted onto tapes over the course of the 7-week sampling period. No
clear diurnal pattern was observed for sporangial dispersal at either site (data not shown).
Rainfall was signiﬁcantly positively correlated (r=0.57, P<0.0001) with detection

of sporangia in the commercial ﬁeld. Rainfall was the only weather parameter that was

7
signiﬁcant in regression analysis (13:3.57, R‘=0.68) for the experimental ﬁeld situation.

Dispersal occurred during 56% (5 of 9) ofdays with rainfall at the commercial farm and
56% (9 of 16) of days with rainfall at the experimental ﬁeld (Figure 1.5). Dispersal
occurred during 42% (5 of 12) of days without rainfall at the commercial farm and 30%
(9 of 30) of days without rainfall at the experimental ﬁeld (Figure 1.5). Vapor pressure
deﬁcit was not signiﬁcant in correlation or regression analysis, respectively for the

commercial or experimental ﬁeld. Wind speed was not measured in the commercial

32

 

10

 

 

 

 

-
CE.
‘I
‘
- .—

 

.3
O

r

 

O A N 9 § a a: N on O
+ Sporangia/m3 air

Y

 

 

 

 

   

 

30 B ............................................. 350
n

25 4 = 300

. ll '5
250

E 20 . " ”a
.. II a
ll 2” "2
I 1 o I
I . 100 +

5 ‘ l 50

0 " . 0

Figure 1.5. Relationship between daily cumulative concentration of airborne P. capsici

sporangia (sporangia/mB/day) and the correspondent daily rainfall (mm) for A,

monitoring in the commercial cucurbit ﬁeld in 2006 and B, monitoring in the

experimental ﬁeld setting in 2008. Note the differences in scale on the y-axes.

33

ﬁeld, and was not found to be signiﬁcant in regression analysis for the experimental ﬁeld.

DISCUSSION

This study suggests that dispersal of P. capsici sporangia via wind currents is
infrequent and that sporangia are unlikely to be dispersed among ﬁelds by wind. This is
in agreement with the conclusions of Lamour and Hausbeck (35,36) from spatiotemporal
genetic diversity data of populations of P. capsici in Michigan and Schlub (46), who
investigated aerial dispersal in a pepper ﬁeld with an active epidemic of Phytophthora
blight.

Direct observations in the laboratory showed that sporangial dispersal occurred in
water with capillary force, but did not occur due to wind or a reduction in relative
humidity. Laboratory results were conﬁrmed in the ﬁeld; volumetric spore sampling
demonstrated aerial dispersal was a low frequency event, especially under commercial
ﬁeld production conditions. Furthermore, a very small amount of the total available
sporangial load was dispersed. In the experimental ﬁeld situation, a total of 438
sporangia were detected from an estimated minimum of 189 to 567 lesions throughout
the 7-week sampling period. In comparison, up to 600,000 sporangia can be dislodged
by water and mechanical force from one lesion with P. capsici sporulating profusely
when a cucumber is inoculated and incubated as described above (data not shown). The
escape of sporangia is limited by the presence of a canopy, and detached sporangia may
be diluted during transport in the wind. These two limitations on detection were
intentionally removed in the experimental ﬁeld setting, but not at the commercial farm.
In cucumber, the plant‘s foliage is typically asymptomatic and sporangia form primarily

on fruit or occasionally at the plant‘s base. Thus, a reduced canopy is not common in

34

cucumber ﬁelds with high disease pressure, and sporangia tend to be low in the canopy
where the wind velocity is reduced as compared to the upper canopy (3). While some
hosts such as pepper may display sporulation on tissue higher in the canopy (43),
sporangia tend to be low in the canopy on the crown and/or fruit tissues of most infected
host plants including acorn squash (29). When we sampled aerial sporangial dispersal
from infected cucumbers placed. on a platform without the limitation of a canopy
(experimental ﬁeld situation) few sporangia were trapped, and often these sporangia were
detected in clusters. Clusters of sporangia should deposit close to the original source due
to the increased settling speed of these clusters as compared to a single sporangium (20).
Based on laboratory and ﬁeld observations from this study, we suggest that aerial
dispersal of sporangia is relatively unimportant for P. capsici.

Unlike what is observed for P. infestans (3) and some downy mildew pathogens
(9,31), atmospheric P. capsici sporangial concentrations did not display a clear diurnal
pattern in either ﬁeld. Only rainfall was statistically associated with dispersal. Dispersal
was sporadic, and sporangial dispersal was not always detected during rain events at
either sampling site. This could be explained by sporangia wash-out during rainy or wet
conditions, or sporangial detachment rates may have been low even when moisture was
present. Our data is supported by a previous study by Schlub (46) that examined aerial
dispersal of P. capsici in a pepper ﬁeld during an active epidemic by using media-ﬁlled
plates on stakes placed at different distances from sporulating host tissue. Schlub
detected dispersal on 5 of 70 sampling occasions. Three of these dispersal events were
associated with rain or overhead irrigation, and the remaining two occurred when plates

were placed within 5 cm of host tissue with actively sporulating P. capsici (46).

35

Irrigation water infested with P. capsici can initiate an epidemic in a ﬁeld lacking
a previous history of infestation (25). Many studies have demonstrated the importance of
water in the development and spread of disease caused by P. capsici (10,40,46). In this
study, we directly observed that sporangia will readily detach and be transported in water
combined with capillary force. It is possible that the observed detachment and
subsequent dispersal could be an artifact of tissue manipulation in setting up the samples.
We maintain that this is unlikely since dispersal was never observed from tissue with P.
capsici sporangia that was subjected to direct wind, although this tissue was manipulated
in a similar fashion. The ability of water to detach and transport sporangia underscores
the importance of water as the primary mechanism of pathogen dispersal.

It is known that wind has the potential to move microbes long distances, either as
spores or in conjunction with soil particles. While it may be tempting to assume that
long-distance atmospheric transport of inoculum is responsible for the sudden appearance
of plant diseases in places far from areas of known pathogen establishment, it also
important to remember that alternative pathways for pathogen transport exist, such as
human-mediated transport (4). Since long-distance dispersal of sporangia via wind is
unlikely, preventing long-distance human-mediated transport of P. capsici via infested
soil and water and infected plant material is key. The current management strategies of
exclusion, cultural control, host resistance, and fungicides and fumigants are still
warranted in light of this study. Growers can circumvent long-distance spread of the
pathogen by cleaning equipment between ﬁelds, not spreading cull fruit onto ﬁelds, and
avoiding the use of surface water that may be contaminated with P. capsici to irrigate

healthy crops (25.29). Water is known to be a key factor in local spread of P. capsici

36

spores and in the development of epidemics (10,40). Water may be managed within a
ﬁeld by planting into well-drained ﬁelds, using raised beds, using black plastic mulch,
trellising cucurbit plants, irrigating sparingly and using trickle irrigation. While it is

important to use a multifaceted approach to manage the diseases caused by P. capsici,

managing water appears paramount in light of the results of this as well as other studies

(29).

37

ACKNOWLEDGMENTS
This research is based upon work supported by the USDA CSREES Special Research
Grant under Award No. 2006-34572-16902; the USDA C SREES Special Research Grant
under Award N0. 2008-34572-19339; the Michigan Agricultural Experiment Station
Hatch Project 01966; and the Pickle and Pepper Research Committee of MSU, Pickle
Packers International, Inc. This study was also supported by a grant from the New York
State Dept. of Agriculture and Markets and the NY State Agricultural Experiment Station
Hatch Project 625-405. We thank P. Zaini, L. De La Fuente, K. Myers, H. Lange, and A.

Camp for their assistance.

38

10.

ll.

12.

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Ferrandino, F. J., and Aylor, D. E. 1984. Settling speed ofclusters of spores.
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snap bean is pathogenic to cucumber fruit and soybean. Abstr. Phytopathology
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Characterization of Phytophthora capsici from Michigan surface irrigation water.
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41

CHAPTER II: EFFECTS OF TEMPERATURE, CONCENTRATION, AGE, AND
ALGAECIDES ON PH YTOPHT HORA CAPSICI ZOOSPORE
INFECTIVITY
ABSTRACT

Granke, L. L. and Hausbeck, M. K. 2010. Effects of temperature, concentration, age,
and algaecides on Phytophthora capsici zoospore infectivity. Plant Dis. 94:54-60.

Controlled laboratory studies were undertaken to determine the effects of water

’) 4 ‘5
temperature (2, 9, 12, 19, 22, and 32°C), spore concentration (1 x 10‘, 1 x 10), 5 x 103, 1

x 104, 2 x 104, and 4 x 104 zoospores/ml), and zoospore suspension age (0, 1, 3, and 5

days old) on infection of pickling cucumbers (Cucumis saiivus) by Phytophthora capsici.
Zoospore motility and mortality in response to commercial algaecides were also
investigated. Cucumbers became infected at all temperatures tested, except 2°C, and the
highest infection incidence was observed for cucumbers incubated in suspensions held at

219°C. Fewer fruit (<40% at 219°C, 0% at S 12°C) became infected. when water

contained 1 x 102 zoospores/ml. Almost 100% of fruit were infected when

water contained 25 x 103 zoospores/ml at temperatures 212°C. While the incidence of

fruit infection declined with the zoospore suspension age, infection still occurred when 5-
day-old suspensions were used. Commercial algaecides inhibited zoospore motility and
caused signiﬁcant zoospore mortality in laboratory assays, and show promise for
treatment of infested irrigation water. Avoidance of infested irrigation water throughout
the growing season is warranted until effective and economically acceptable water

treatments are developed for ﬁeld use.

42

INTRODUCTION

Michigan is the largest producer of pickling cucumbers (Cucumis sativus) in the
United States (3). The oomycete plant pathogen Phyophthora capsici Leonian has a
large host range including vegetable crops in the C ucurbitaceae and Solanaceae (16).
Cucumber fruit are especially susceptible to infection even though infected cucumber
plants may be relatively asymptomatic. In Michigan, entire ﬁelds of cucumbers bearing
mature fruit, and semi truck loads of harvested fruit may be rejected due to fruit rot (22).
P. capsici may generate copious amounts of asexually produced sporangia on the host
surface after infection has been successfully established. Mature sporangia may
germinate directly, forming one to several germ tubes or, when in contact with free water,
will differentiate into 20 to 40 biﬂagellate motile zoospores (22). Phytophthora
zoospores may swim electro-(SO) and chemotactically or may be carried in ﬂowing water
(41), including irrigation water. Once a zoospore comes in contact with host tissue, it
encysts, attaches, germinates by forming a germ tube, and penetration and colonization of
the host tissue follows (16).

Phytophthora spp., including P. capsici, have been detected in surface water used
for irrigation in diverse geographic regions of the United States (19,37,42). It has been
hypothesized that irrigation water infested with P. capsici may initiate a disease epidemic
of susceptible host plants within a ﬁeld lacking a previous history of infestation (19).
Zoospores are known to be the primary propagules found in irrigation water (10,46), but
the ability of zoospores to cause infection over time at different inoculum densities has
not been determined. In a study in Florida. P. capsici propagules could be detected for at

least 45 days following incubation in tailwater ponds in ﬁne pore membrane covered-

43

. 5 . .
bottles that contained ~1.5 x 10 zoospores suspended in tailwater, surface runoff water

that is collected in retaining ponds and canals (42).

While the optimal temperature for growth of P. capsici has been described
(16), the effect of water temperature on infection of cucumbers by zoospores is unclear.
Studies in Florida did not ﬁnd a correlation between temperature and detection of P.
capsici propagules in tailwater (42), but studies in Michigan did ﬁnd a signiﬁcant
correlation between temperature and detection of P. capsici in surface water irrigation
sources (19). Understanding the relationship between water temperature and the ability
of zoospores in water to infect cucumber fruits is required for successful management of
disease when irrigation water may be infested.

Most growers in Michigan utilize surface water to irrigate vegetable crops since
well water is more expensive and less available. Michigan cucurbit growers provide an
average of 2.5 cm of water per week to their crops during the summer growing months.
Gevens et al. (19) found that the detection of P. capsici in surface water irrigation sources
was greatest during July and August when the need to irrigate crops was also the greatest.

The increasingly common greenhouse/nursery practices of recycling irrigation
water and recirculating nutrient solutions are economically and environmentally
beneﬁcial, but also increase the spread of waterborne pathogens including P. capsici
(46,47). There are several methods currently available for disinfesting contaminated
irrigation water in greenhouse and nursery settings including the use of antimicrobial
compounds such as copper (48,49) and hydrogen peroxide (23,43), but little research has

focused on disinfestants for use in a ﬁeld setting.

Commercial algaecides contain copper sulfate, chelated copper, or sodium
carbonate peroxyhydrate as the active ingredient. It has long been known that copper is
suppressive to Phytophthora spp. (21,45), and spray tank-mixing copper with other
fungicides has been shown to improve management of P. capsici in the ﬁeld (22).
Previous studies have shown that increasing copper ion concentrations in nutrient
solutions reduces disease caused by P. cinnamomi (49) or P. cryptogea (48), and recent
laboratory studies have shown copper-based algaecides have potential for treating P.
ramorum in water (1 1). Sodium carbonate peroxyhydrate (SCP)-based algaecides were
developed as a nonpersistent alternative to copper algaecides (27,39), which may build up
in aquatic systems and can be toxic to nontarget organisms (30,34). SCP releases
hydrogen peroxide upon dissolution in water (53), a compound that is active against a
wide variety of organisms ( 28) and is an effective treatment to control oomycete parasitic
infection of ﬁsh eggs (5.27). Chlorination and algaecide treatments are already used by
some growers to control undesirable algal growth in irrigation systems (36) since algae
may serve as a substrate for undesirable bacterial growth and may clog intake screens and
ﬁlters and other system components, resulting in reduced water ﬂow rates and an
inadequate supply of water for irrigated crops (13.35).

The objectives of our study were to elucidate the effects of: (i) water temperature,
inoculum concentration, and zoospore suspension age on infection of pickling cucumbers
by P. capsici and (ii) copper and SC P-based algaecides on zoospore motility and

mortality.

45

MATERIALS AND METHODS
P. capsici isolates and production of zoospore inoculum. Actively growing
cultures of P. capsici isolates SP98 and OP97 were used (isolate notation refers to the
culture collection maintained in the laboratory of M. K. Hausbeck at Michigan State
University). The cultures were derived from long-term stock cultures (stored at 20°C in
sterile 1.5-ml microcentrifuge tubes with 1 ml of sterile water and one sterile hemp seed)

that were transferred to unclariﬁed V8 agar (UCV8, 160 ml unclariﬁed V8 juice, 840 ml

distilled water, 30 mM CaCO3, and 1.5% agar). Isolates were maintained on UCV8 at

room temperature (21 : 2°C) under continuous fluorescent lighting and were transferred
to new UCV8 weekly. OP97 (A1 mating type, sensitive to mefenoxam) was isolated
from cucumber in Michigan and was used for the studies looking at the effects of water
temperature, inoculum concentration, and zoospore suspension age on infection of
pickling cucumber fruits by P. capsici. SP98 (A2 mating type, sensitive to mefenoxam)
was isolated from pumpkin (Cucurbita pepo) in Michigan and was used for the studies
examining the effects of copper and SCP-based algaecides on zoospore motility and
mortality. Studies by Gevens et a1. (1 8) demonstrated that SP98 and OP97 are equally
virulent on cucumber fruits. Zoospore suspensions were prepared by ﬂooding actively
sporulating P. capsici cultures with distilled water and incubating the plates at 2°C for 1
h followed by 30 min at room temperature. The concentration of zoospores was
determined using a hemacytometer and adjusted to the desired concentration using
distilled water at the appropriate experimental temperature.

The effect of water temperature and inoculum concentration on infection.

Six temperatures (2, 9, 12, 19, 22, and 32 ﬂ: 2°C) and six inoculum densities (1 x 102, 1 x

2 3 4 . .
103, 5 x 10), 1 x 10 , 2 x 104, and 4 x 104 zoospores/ml) were tested to investigate the

effects of water temperature and inoculum concentration on infection of pickling
cucumbers. Pickling cucumber fruits ofa similar size (10 :1: 1 cm long) were purchased
from a local supplier. For each temperature/concentration pair, six detached unwounded
pickling cucumbers were rinsed to remove surface debris, surface disinfested with a 10%
bleach (0.62% NaClO) solution for 5 min, air dried, and placed into 1 1.4-1iter plastic tubs .2
containing 3 liters of P. capsici isolate OP97 zoospore suspension. The fruits were kept _

in the zoosopore suspension for 24 h at the desired temperature, rinsed with distilled

 

water, and incubated in a moist chamber at ~100% relative humidity overnight at room

rm”. - - -

temperature. When lesions with water soaking and/or pathogen growth were apparent on
fruits, tissue sections from the margin of lesions were plated onto UCV8 plates amended
with 25 ppm of benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of
pentachloronitrobenzene (BARP). When fruits appeared healthy, at least three tissue
sections were randomly excised from the fruit and plated onto BARF-amended UCV8.
Plates were incubated at room temperature under continuous ﬂuorescent lighting for 3 to
5 days and were observed using a compound microscope. P. capsici was conﬁrmed
using morphological characteristics according to the Phytophthora spp. key by
Waterhouse (52). The resulting P. capsici colonies were transferred to new UCV8, and
about 96% of isolates were characterized for mating type and mefenoxam sensitivity (26)
to conﬁrm they were the same as OP97. Six control cucumbers were added to 3 liters of
distilled water at room temperature and treated as above to ensure that the cucumber
fruits used in the experiment were not infected with P. capsici at the onset of the

experiment. The temperatures ofthe zoospore suspensions were monitored throughout

47

the course of the experiment using Watchdog 100 series temperature loggers (Spectrum
Technologies, Plainﬁeld, IL). Three independent experiments were conducted for each
temperature/concentration combination.

The effect of zoospore suspension age on infection of pickling cucumber
fruits. The effect of zoospore suspension age on infection of pickling cucumber fruits

was tested by adding six unwounded pickling cucumbers to 3 liters of OP97 zoospore

suspension for each inoculum concentration tested (1 x 102, 1 x 103, 5 x 10), l x 104, 2

x 104, and 4 x 104 zoospores/ml) at 0 (immediately), 1, 3, or 5 days following zoospore

suspension creation. Cucumbers were removed 24 h later and treated as described above.
Six cucumbers were added on day 0 to 3 liters of distilled water and treated as the other
fruits for a control. All experiments were conducted at room temperature. Four
independent experiments were conducted for each age/inoculum concentration
combination.

The effects of algaecides on zoospore motility and mortality. Commercially
available algaecides were assessed for effects on zoospore motility and mortality in
water. The algaecides tested contained either chelated copper or SCP as the active
ingredient and were used at rates allowable in surface water bodies in Michigan (2)
(Table 2.1). Fresh stock solutions containing twice the desired rate of algaecides were
created immediately prior to experiments. A bleach stock solution containing ~4 ppm
chlorine was used to treat zoospores in water at ~2 ppm chlorine as a positive control
(24). Distilled water was used as a negative control. The pH of algaecide-treated water
was measured three times using a 820 pH meter with InLab Expert Pro electrode

(Mettler-Toledo, Columbus, OH).

48

Table 2.1. Algaecide treatments applied to water containing Phytophthora capsici isolate

SP98 zoospores and the resulting pH of the water.

 

Treatment

 

. . . Z
(pro duct/liter) Active ingredient (% ) pHi SEy
8196:?) sodium hypochlorite (6.2) 7'61i0'01
Cutrine-Plus . 8.55i0.02
(1 8 ml) copper ethanolamine (9)

Cuggiréer-nliltra copper ethanolamine (9) 7'87i0'03
Szlzmlmnigy copper triethanolamine and copper hydroxide (8) 7'16i0'01
(C12???) copper carbonate (9.1) 8'71i0'01
GreenClean . . 987220.00

(0.006 mg) sodium carbonate peroxy hydrate (50)
(gig?) copper triethanolamine and copper hydroxide (8) 7°64i0°01
1:02???an 30 copper sulfate pentahydrate (2.7) 6'34i0'16
Pak 27 . 9.572t0.00

(0.006 mg) sodium carbonate peroxyhydrate (85)
EarthTec (1 ml) copper sulfate pentahydrate (5) 6.21i0.03
Greepregp Pro sodium carbonate peroxyhydrate (85) 9'57i0'00
Alg18y3C1rE§WF copper citrate and copper gluconate (5) 5'70i0'02
Distilled Water 6.67i0.01

None

 

Z . .
Copper (%) IS expressed as elemental copper equivalent

y SE=standard error

49

For the zoospore motility experiments. 5 ml of stock solution or distilled water

were mixed with 5 ml of distilled water containing 1 x 106 zoospores/ml (isolate SP98)

in a 60 x 15 mm plastic Petri dish. The treated suspension was checked using a
compound microscope every 3 min for the ﬁrst 15 min, then every 15 min until 1 h after
treatment, and then every 30 min thereafter until no zoospore movement was observed.
The amount of time required for all zoospores to cease movement was recorded for three
replicate plates of each treatment. The experiment was conducted three times.

Zoospore mortality was assessed after treatment for 5, 10, 30, 60, or 120 min.

Algaecide and control treatments were initiated by mixing 1 ml of stock solution or water

with 1 ml ofa suspension containing 1 x 104 zoospores/ml (isolate SP98) in a 2.2-ml

microcentrifuge tube. After the desired contact time was reached, the tube was vortexed
brieﬂy, and three 100-ul aliquots of treated zoospore suspension were diluted with 100
ml of distilled water each in 125-ml glass Erlenmeyer ﬂasks to stop the reaction. The
resulting treated zoospore suspension was vacuum ﬁltered through quantitative ﬁlter
paper with l-um particle retention (VWR, Batavia, IL), and the ﬁlter paper was plated

onto BARF-amended CV8 (160 ml clariﬁed V8 juice. 840 ml distilled water, 30 mM

CaCO3, and 1.5% agar). The plates were incubated at room temperature under

continuous ﬂuorescent lighting for 48 h. The ﬁlter papers were removed, colonies were
enumerated, and ~50% ofcolonies were observed via compound microscopy to conﬁrm
P. capsici using morphological characteristics according to the Phytophthora spp. key by

Waterhouse (52). Zoospore mortality was calculated by comparing the number of

50

 

colonies that formed from treated suspensions to the number of colonies from the
untreated control. The experiment was conducted twice.

Data Analyses. All statistical analyses were performed using the SAS statistical
package version 9.1 (SAS Institute, Inc., Cary, NC). Prior to analysis, percentage data
were normalized by angular (arc sin) transformation. Data were subjected to analysis of
variance (P5005), and Fisher’s protected LSD was used for separation of means
(P5005). Unequal variance models were used to evaluate the effects of temperature and
zoospore concentration on infection incidence, zoospore suspension age and zoospore
concentration on infection incidence, and algaecide treatment and contact time on
zoospore mortality. Variances were grouped by the concentration factor, age factor, and
treatment factor, respectively. Data from 2°C were removed prior to analysis since no
infection occurred at this temperature. Regression analysis was used to determine the
relationship between each variable and mean infection incidence. The model that best ﬁt
the data was used. The relationship between temperature and infection incidence was
analyzed using nonlinear polynomial regression analysis. The relationship between
zoospore inoculum concentration and infection incidence was analyzed using logarithmic
regression analysis. The relationship between zoospore suspension age and infection
incidence was determined using linear regression analysis after data were subjected to
analysis of covariance to determine if the same slope could be used for all of the data

despite differences in zoospore suspension concentrations.

51

RESULTS

No infection occurred when cucumbers were incubated at 2°C regardless of
zoospore inoculum concentration. Cucumber fruits became infected with P. capsici at all
other temperatures tested (Figure 2.1). A statistically similar infection incidence
(P>0.41) was observed for fruit incubated in suspensions at 219°C. The results showed a

strong positive relationship between water temperature and cucumber fruit infection

. . . . . 2

ineldence when analyzed usmg a second order polynomial regressron (y=-0.2x +7.6x-
2 . . . 3 4 4

9.8, R =0.96). When a suspensmn containing 1 x 10 , 2 x 10 , or4 x 10 zoospores/ml

was used, temperature did not signiﬁcantly affect infection incidence. However,

temperature did signiﬁcantly affect infection incidence for all other zoospore

concentrations tested. When a suspension containing 1 x 102 zoospores/ml was used,
fewer fruit became infected than at any other concentration tested (P<0.0008). When a
suspension concentration of 1 x 103 zoospores/ml was tested, fewer fruit became infected
than at higher densities (P<0.0002). Zoospore suspensions containing 5 x 103 or 1 x 104

zoospores/ml resulted in similar levels ofinfection (P=0.1479); suspensions containing

21 x 104 zoospores/ml also resulted in similar levels of infection (P>0.28). The results

indicate a strong positive relationship between inoculum concentration and cucumber

fruit infection when analyzed using logarithmic regression (y=15.9Ln(x)-61.7, R2=0.97).

. . . . 4 4
Overall, suspensrons With concentrations from 1 x 10 to 4 x 10 zoospores/ml caused

the greatest amount of fruit infection at all temperatures tested (Figure 2.1). A signiﬁcant

52

 

 

 

m bdays

 

 

I
..///////////////////////////////////

 

 

woman

0
2
1

SSS

yyy

aaa

ddd

135

I

V .
o o o w o o
AOI 8 6 2

3.. 8.8.... «:5.

 

404-4

 

Sci-3

10+3

0+2

Concentration (zoosporeslml)

Figure 2.1. Effects of Phytophthora capsici zoospore inoculum concentration and A,

temperature (data from 2°C not shown as no infection was observed), and B, zoospore

age on infection of cucumbers. Each column represents the average of three (A) or four

(B) repeated tests with six replicate pickling cucumbers per treatment per test. Error bars

- represent standard error.

53

interaction between temperature and inoculum concentration was noted.
A strong negative relationship between zoospore suspension age and fruit

infection incidence was noted when linear regression analysis was applied (y=-

2 . . . .
1 1.0x+82.0, R =0.96). More infection occurred when zoospore suspenSionS were used
immediately after creation than when 1, 3, or 5-day-old suspensions were used

(P<0.0016). The amount of infection was similar for 1 and 3-day-old suspensions

(P=0.3277), but not for l and 5-day-old suspensions (P=0.0005) or for 3 and 5-day-old

,
suspensions (P=0.0244). Suspensions containing 1 x 10“ zoospores/ml caused

signiﬁcantly less infection than any of the other densities tested (P<0.0094). Suspensions

containing 1 x 103 zoospores/ml caused signiﬁcantly less infection than suspensions

. . 4 4 . . . .
containing 1 x 10 to 4 x 10 zoospores/ml (P<0.0279) and Similar amounts of infection
to suspensions containing 5 x 10) zoospores/ml (P=0.0575). Suspensions containing 25

x 10 zoospores/ml caused statistically Similar amounts of infection (P>0.47, Figure

2.18). There was no signiﬁcant interaction between zoospore suspension age and
concentration. All of the isolates recovered from the inoculated cucumbers at the end of
experiments were found to have an A1 mating type and were sensitive to mefenoxam
(data not shown).

All of the algaecides tested caused zoospore swimming to cease within three min
of treatment (data not shown). The corresponding control zoospores were motile for 4.5
to 6 h. Algaecide treatments caused Signiﬁcant mortality of zoospores in water (Figure

2.2). A contact time of 230 min resulted in greater zoospore mortality than a contact time

54

of 5 or 10 min (P<0.0039). Overall, statistically similar zoospore mortality was observed
after treatment for 30, 60, or 120 min (P>0.69). Several of the copper-based and one of
the SPC-based algaecides showed efficacy similar to bleach, the positive control, in
killing zoospores when the contact time was 230 min (P>0.10, Figure 2.2), and all of
treatments were better than the negative control (P<0.0001). There was no signiﬁcant
interaction between the algaecide treatment and contact time. The water was more
alkaline following treatment with most (8 of 1 1) of the algaecides tested and bleach

(Table 2.1).

DISCUSSION

It is important that use of infested irrigation water is avoided to prevent the
introduction of P. capsici to uninfested agricultural land, or that effective treatments are
found so that water sources may be disinfested and safely used to irrigate susceptible
crops. This study found that the incidence of cucumber fruit infection was greater with
increasing temperature and inoculum concentration and lower with increasing age of the
zoospore suspension. Upon treatment with copper or SC P-based algaecides at rates
allowable for Michigan surface waters, zoospore motility was inhibited and signiﬁcant
zoospore mortality was noted.

Zoospores in water at 9 to 32°C were able to infect pickling cucumber fruits, yet
no infection occurred at 2°C. Since zoospores in water at 9°C were able to infect fruits,
hydrocooling water, which is usually around 10°C (14), may be a source of postharvest
P. capsici inoculum, but this has not been investigated to date. Hydrocooling water is

often recycled, which may lead to a buildup of pathogens in the water (51), but chlorine

55

 

 

- 10 min ‘
m 120 min

 

 

.*w%///////////////////V///////V////ﬂ///////é

 

.*7////7////%///////////%/////////////////IIM

 

Tug/dag;

Eng/gig

 

.*w/V/////r////////ﬂﬂﬂ/IV///.ﬂﬂ/IV/////ﬂﬂ///ﬂﬂ¢

.*7////////V////////////////////////V/////%

 

 

- b b P 1P
0 o o o o
o w

2

8 6
1 ex; Eire... SonnOON

Figure 2.2. Effects of algaecide treatment and contact time on Phytophthora capsici

zoospore mortality (%). Each column represents the average of two repeated tests with

three replicate culture plates per test. Error bars represent standard error. Columns with

an asterisk are not signiﬁcantly different from the positive control, bleach, at 230 min

contact time (LSD, P>0.10).

56

treatment may be used to limit pathogen spread (17). Chlorine also limits survival of P.
capsici zoospores (24), and future studies are necessary to elucidate the role of
hydrocooling water in postharvest infection of cucumbers.

Washing pits are another potential source of postharvest inoculum that has not
been characterized to date, and future studies should investigate whether or not washing
pits are important for postharvest infection of fruit. In Michigan, most cucumbers are
harvested mechanically into semi trucks for transport to the processing facility, where
they are dropped into a washing pit. Washing pits are usually ﬁlled with cold well water
in the morning and the water warms throughout the day to ambient temperatures
(Hausbeck, personal communication). Since both water temperature and possibly the
concentration of inoculum in the water increases throughout the day as multiple loads of
cucumbers are washed in the same pit, these washing pits could potentially be responsible
for postharvest infection of washed fruits. The results of our study Show that increased
temperature and inoculum densities favor zoospore infection of fruits. Furthermore,
during harvest and handling, cucumber fruits may be dropped distances of >3 m, which
constitutes a major source of mechanical injury for the fruit (32). While mature
cucumber fruits are less susceptible to infection by P. capsici (18), wounding has been
shown to negate age-related resistance in pepper (7), and wounded cucumbers may be
more susceptible to infection.

A greater percentage of fruits were infected following incubation in zoospore
suspensions held at temperatures 219°C in our study; this was especially apparent when
water contained a lower concentration of zoospores. A Si gniﬁcant interaction was found

between temperature and inoculum concentration in our research. A positive correlation

57

‘_H

 

I‘L: - .-I' D .

between water temperatures and infection incidence was observed in our study, which
agrees with previous studies on P. capsici (19) and P. cinnamomi (37). Biles et al. (6)
found that 27°C was optimal for zoospore infection of pepper fruits, and fruits were not
infected at 15 or 35°C in laboratory studies, which is a more limited range than was
found in our study. Biles et al. used a different isolate and host from that used in our
study, which may explain some of the variation observed. A large volume of inoculum
was used for our experiment, which may have increased the potential for infection (6).
Previous research with P. iiy’estans Showed that zoospores are more susceptible to
unfavorable environmental conditions than sporangia, and zoospore survival is negatively
affected by cool temperatures (3 8). Gevens et a1. (19) did not detect P. capsici in surface
water irrigation sources via baiting (infection of pear and cucumber fruits) when the
water temperature was <14°C or >25°C. It is likely that the concentration of zoospores in
surface water sources such as ponds or rivers is lower during much of the growing season
than the densities used in laboratory studies, and zoospores may be absent from irrigation
surface water sources for part of the growing season. In the ﬁeld, additional factors may
inﬂuence zoospore motility and infectivity such as solar radiation, pH, ion, amino acid,
sugar, and salt concentrations (9,16,31) and parasitism. Survival of P. infestans
zoospores was strongly affected by ultraviolet radiation in previous studies (31,38).
While solar ultraviolet radiation declines exponentially with water depth (8), motile
Phytophthora zoospores display negative geotropism placing them toward the surface of
a water body (9).

Our study found zoospore inoculum concentration Signiﬁcantly affected infection

incidence of pickling cucumbers. There appeared to be a threshold at 1 x 104

58

zoospores/ml; increasing the concentration beyond this did not result in a greater amount

of infection at most of the temperatures tested. This is Similar to previous research in

. . . 4 .
pepper that showed. that a suspenSion containing 21 x 10 P. capscz zoospores/ml

resulted in 100% infection of susceptible plants at normal greenhouse temperatures (40).

In agreement with a previous study using P. nicotianae (4), signiﬁcantly greater levels of

. . . . . . 4
disease were observed With increaSing inoculum concentration up to 1 x 10

zoospores/ml. However, it is unlikely that 1 x 104 zoospores/ml would be present in

irrigation water, so it signiﬁcant to note that at temperatures 219°C, zoospore suspensions

containing 1 x 102 zoospores/ml, the lowest concentration tested, resulted in infection of
cucumbers.

Mean survival of zoospores of several Phytophthora spp. (not including P.
capsici) in sterile distilled water or ﬁltered lake water declined to <20% in 48 h in a
previous study (10). Shokes and McCarter found that zoospores of P. aphanidermatum
died rapidly, with only 4.2% surviving at day 6 and 0% at day 12 (44). In this study, it

was found that the infection incidence of cucumber fruits was signiﬁcantly reduced when
. . . 3

older zoospore suspenSionS were used. When 5-day-old suspenSions With 51 x 10

zoospores/ml were used, no fruit became infected, and few fruit were infected for 5-day-

old suspensions containing 5 x 103 zoospores/ml. Since it is thought that P. capsici-

infested water may initiate an epidemic in a ﬁeld without a previous history of infestation
when planted to a susceptible host (19), it is signiﬁcant that zoospores can survive and

initiate infection for 5 days. Furthermore, complete resistance to P. capsici has not yet

59

been observed for any cucumber cultivar, although reduced pathogen sporulation has
been observed on some varieties (18). Roberts et al found zoospores were able to survive
for 45 days in tailwater in Florida (42). Neither our study nor the Florida study
investigated whether encysted zoospores were the surviving propagules or if the
zoospores had germinated to produce mycelial fragments that survived.

Our research suggests that algaecides may be a viable disinfestant for P. capsici-
contaminated irrigation water. All algaecides tested caused a signiﬁcant reduction in
zoospore motility and mortality. Previous work has Shown zoospores are able to remain
motile longer at higher cell densities (20). All zoospores ceased to swim after 53 minutes
of exposure to algaecide in our experiments, despite the high zoospore concentration
used. In addition to greatly reduced motility, signiﬁcant mortality of zoospores was
observed after algaecide treatment in our study. Copper-based algaecides also caused
signiﬁcant mortality of P. ramorum zoospores in another recent study (11).

Treatment ofthe water with algaecides modiﬁed the pH of the water, usually
resulting in an increase in alkalinity. Recently, Kong et a1. (25) found that survival of
zoospores of various Phytophthora spp. was affected by the pH of the solution in which
they had been incubated. The optimal pH for zoospore survival varied by species from
pH 5 to 9, and survival was generally poor at pH (3 or 11) extremes. Furthermore, P.
palmivora zoospores in solutions with pH 6 or 8 encysted twice as quickly when
compared to zoospores kept in solutions with pH 7 (33). While P. capsici was not
included in either of these studies, it is likely that zoospores of P. capsici in water are
affected by pH in a manner similar to other Phytophthora spp. Commercial algaecides

used to treat algae in irrigation water sources may also be useful to reduce P. capsici

60

 

propagules, but more research is needed before recommendations can be made. Since
algaecides are already used to prevent algae growth and clogging of irrigation equipment
(36), this treatment option would not result in additional management costs for many
growers.

Further work is necessary to investigate the efﬁcacy of algaecides in ﬁeld and
greenhouse settings and to explore potential problems with using algaecide-treated water
for irrigation, such as phytotoxicity in plants and effects on nontarget species such as ﬁsh.
The effectiveness of copper-based a1 gaecides is affected by the concentration and form of
copper applied, the duration of exposure, and characteristics of the water body (34). A
copper half-life of 2.6 to 5.7 days was observed in a previous study when algaecides were
applied to barrels containing water and sediment with different physical characteristics
(34), and elevated copper concentrations were observed for several weeks following
algaecide treatment of a reservoir (15). The longest half-life was observed when low
hardness, low alkalinity water was used in the barrel experiments (34). The persistence
and concentration of copper in water and sediments Signiﬁcantly affects the impact on
nontarget species including phytoplankton and ﬁsh (30), and this Should be considered if
algaecides are applied to a water source to reduce the number of P. capsici zoospores.
Excess copper in irrigation water may lead to phytotoxicity including reduced plant
growth and photosynthetic ability (1). Sodium carbonate peroxyhydrate has been less
well studied than copper, but it is known that efﬁcacy is affected by the soluble organic
matter content of the water body (29). Hydrogen peroxide may be phytotoxic, especially

to young plants, when used at rates required to control algae (12). Future studies should

61

focus on investigating the efﬁcacy and potential phytotoxic effects of algaecides in ﬁeld,
greenhouse, and processing (postharvest treatment) settings.

In summary, zoospores are able to infect cucumber fruits at a wide range of
ecologically relevant water temperatures, which suggests that avoidance of P. capsici-
infested water is necessary throughout the entire growing season. The emergence of new
treatment options such as algaecides may provide easy, economically acceptable options
for disinfestation of surface water irrigation sources in the future, but further research is

warranted to investigate the efﬁcacy of these products in the ﬁeld setting.

62

ACKNOWLEDGMENTS
This research is based upon work supported by the USDA CSREES Special Research
Grant Program under Awards No: 2006-34572-16902 and 2008-34572-19339; the
Michigan Agricultural Experiment Station Hatch Project 01966; and the Pickle and
Pepper Research Committee of MSU, Pickle Packers International, Inc. We thank K.
Heslip for technical assistance. W. Wang for help with statistical analyses, S. Linderman
for editing ﬁgures and tables, and L. Quesada-Ocampo, S. Windstam, and B. Webster for

critical review of the manuscript.

63

10.

11.

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68

CHAPTER III: EFFECTS OF TEMPERATURE, HUMIDITY, AND WOUNDING
ON DEVELOPMENT OF PHYTOPHTHORA ROT OF CUCUMBER
FRUIT.
ABSTRACT

The effects of temperature (10, 15, 20, 25, 30, and 35°C) and relative humidity
(~35, 60. 70, 80, and 100%) on development of Phytophthora fruit rot of pickling
cucumber were investigated in controlled growth chamber studies. The effect of
wounding on disease development was characterized for small (2.0 to 2.5 x 8 to 9 cm),
medium (3.0 to 4.0 x 12.0 to 13.0 cm), and large (>4.5 cm x >14 cm) pickling
cucumbers. Lesions developed on cucumbers incubated at all temperatures tested, except
10 and 35°C. Disease severity was greatest on cucumbers incubated at 25°C at 4 days
post inoculation (dpi). Lesions formed on cucumbers incubated at all relative humidities
tested. The diameter of water-soaking and pathogen growth was greater under conditions
of higher relative humidity. Wounding lessened size-related resistance in pickling
cucumber. The diameter of water-soaking was similar for all wounded cucumbers at 4
dpi regardless of fruit size. While sporangia were formed on wounded large fruits, more
sporangial production was observed in lesions on small and medium fruits. These results

indicate Phytophthora capsici is capable of infecting cucumbers under a wide range of

temperature and relative humidity conditions.

69

INTRODUCTION

The oomycete plant pathogen Phytophthora capsici Leonian causes fruit rot,
foliar blight, and crown and root rot of many vegetable crops in the Cucurbitaceae and
Solanaceae (9), as well as snap and lima bean (Phaseolus vulgaris L. , Phaseolus lunatus
L.) (5.1 l) and Fraser fir (Abiesgfraseri (Pursh) Poir.) (18). Fruit rot of cucumber
(Cucumis sativus L.) is common when weather is favorable for disease development and
P. capsici is present (15). Michigan is the largest producer of pickling cucumbers in the
United States (2), and many of the major growing areas of the state are affected by P.
capsici. Entire ﬁelds ofmature cucumber fruits and semi truck loads of harvested
cucumbers may be lost due to fruit rot (15).

Climatic conditions inﬂuence development of diseases caused by Phytophthora
spp. (8). The optimal growth temperature for P. capsici in culture media ranges from 24
to 33°C (17). Reports of minimum and maximum growth temperatures vary, but range
from 6 t012°C and 32 to >35°C, respectively (9,20,21). Temperature effects on infection
of detached pepper leaves by P. capsici were previously described; a greater percentage
of leaves were infected following incubation at 23°C as compared to 15 or 31°C when the
sporangial suspension was removed from the leaves 4 hours post inoculation (19). If the
suspension was left on the leaves for 8 or 24 hours post inoculation, disease incidence
was similar for leaves incubated at 23 or 31°C (19). Disease incidence and lesion length
were greatest when inoculated pepper fruits were incubated at 27°C in a previous study
(4).

A number of factors including temperature (7), nutrition (23), light (14),

availability of sterols (16), and relative humidity (15,19) are known to affect P. capsici

70

sporangial production. Sporangial production is optimal at 24 to 27°C (7). Lamour and
Hausbeck (15) suggested that sporangial production on slicing cucumber fruits was
greater at 60 and 80% relative humidity than at >90%. Sporangial production following
incubation at 25°C was greater for pepper stem pieces that were incubated at 100 or
97.5% relative humidity than at 92.5 or 87% relative humidity (19).

Previous work has shown that fruit age and size are important factors for
symptom severity and sporangial production on cucumbers (12) and peppers (3). Gevens
et a1. (12) found that oversized pickling cucumbers with a diameter >4.5 cm and length
>14 cm were more often symptom-free following inoculation than smaller fruits.

Pickling cucumbers with a diameter 23.5 cm had much lower disease ratings than smaller
fruits in the same study. Size-related resistance was also observed for a slicing cucumber
variety, with more water-soaking and sporulation being observed on the smallest fruits
tested (12). A similar phenomenon has been observed for pepper fruits, where
unwounded young pepper fruits became more severely diseased than mature pepper fruits
(3). However, when the peppers were wounded, lesion expansion was Similar for all
fruits, regardless of age (3). It is currently unknown whether or not wounding is able to
negate age-related resistance in cucumber.

The objective of our study was to elucidate the effects of temperature, relative
humidity, and wounding on development of Phytophthora rot of cucumber and on

pathogen growth and sporulation on infected cucumber fruits.

MATERIALS AND METHODS

Generation of host and pathogen material. Cucumber fruits (‘Vlaspik’) were

grown at the Michigan State University (MSU) Plant Pathology F arm, East Lansing, MI,

71

a Site without a history of P. capsici. Cucumber plants were treated weekly with either
propamocarb hydrochloride (Previcur Flex, Bayer CropScience, NC) or chlorothalonil
(Bravo Weather Stik, Syngenta Crop Protection, CA) to control downy and powdery
mildews and were fertilized and watered according to standard production practices. For
the experiments investigating the effects of temperature and relative humidity on disease
development, fruits were hand-harvested when 2 to 3 cm in diameter, washed in a 2%
bleach (0.12% NaClO) solution, rinsed, surface disinfested with a 20% bleach (1.24%
NaClO) solution for 5 min, rinsed, air dried, and placed into the incubation chambers
prior to inoculation. Incubation chambers consisted of clear polystyrene boxes with lids
(23 x 10 x 32 cm, Potomac Display, Hampstead, MD).

For the wounding experiments, cucumbers were hand-harvested at three sizes:
small (2.0 to 2.5 cm diameter x 8 to 9 cm long), medium (3 to 4 cm diameter x 12 to 13
cm long), and large (>4.5 cm diameter x >14 cm long). Fruit Size was chosen based on
the work of Gevens et al. (12) to represent different levels of age/size-related resistance
to P. capsici. Cucumbers were washed and surface disinfested as described above, and
placed into moist chambers prior to treatment and inoculation. Moist chambers consisted
of aluminum trays containing wet paper towels, which were sealed using plastic ﬁlm and
tape following inoculation.

Actively growing cultures of P. capsici isolates SP98 and OP97 were used
(isolate notation refers to the culture collection maintained in the laboratory of M. K.
Hausbeck at MSU). The cultures were derived from long-term stock cultures and passed

through cucumber fruits according to the methods of Quesada—Ocampo et a1. (18) before

72

 

use. Isolates were maintained and zoospore suspensions created as described previously
(13).

Effects of temperature on disease development. Two incubation chambers
were placed into a growth chamber set at 10, 15, 20, 25, 30, or 35°C (16/8 day/night
photoperiod and ~95 mE of light intensity). Air from a TopFin XP-125 aquarium pump
(PetSmart, Phoenix, AZ) was humidiﬁed by pumping it through an aquarium airstone
submerged in 1.5 liters of distilled water in a 2-liter Erlenmeyer ﬁlter ﬂask. Air exiting
the ﬂask was routed to the incubation chambers using clear tygon tubing. A small piece
of wet paper towel was added to each box to ensure high relative humidity (~100%) was
maintained. Air exited through a 6.4-mm hole that was drilled into the opposite side of
the chamber lid.

For each temperature treatment, 24 fruits were used (12 per incubation chamber).
Each fruit was inoculated with a single lO-ul droplet of zoospore suspension (isolate
SP98). A Single IO-ul droplet of water was placed on each of six control cucumbers that
were incubated at room temperature (~21 i: 2°C) and high relative humidity to ensure that
the cucumber fruits used in the experiment were not infected with P. capsici at the onset
of the experiment. Six fruits were chosen arbitrarily and removed from each temperature
treatment at 1, 2, 3, and 4 days post inoculation (dpi) for evaluation.

Each fruit was evaluated for water-soaked lesion diameter (visible dark
discoloration of the fruit surface) and the diameter of pathogen growth in the lesion by
taking 2 perpendicular measurements of each lesion for each parameter. The number of
sporangia in each lesion was determined as follows. If sporangia could be observed in a

lesion via stereomicroscopy (Leica M165C, Wetzlar, Germany), the area of pathogen

73

growth was carefully excised with a razor blade and placed into 1 ml of water in a 2.2-ml
microcentrifuge tube. The tube was vortexed for 70 5, plant material removed using
forceps, and number of sporangia in the tube estimated using a hemacytometer. If
sporangia could be observed via stereomicroscopy, but could not be detected via the
method above, the sporangial suspension was concentrated by centrifuging the tube for 5
min at 14,000 rpm. The supernatant was removed and the sporangia were re-suspended
in IOO-ul of distilled water and numbers of sporangia estimated using a hemacytometer.
When lesions were apparent on fruits but no sporangia observed, tissue sections

from the margin of each lesion were plated onto UCV8 (160 ml unclariﬁed V8 juice, 840

ml distilled water, 30 mM CaCO3, and 1.5% agar) plates amended with 25 ppm of

benomyl, 100 ppm of ampicillin, 30 ppm of rifampicin, and 100 ppm of
pentachloronitrobenzene (BARP) to conﬁrm infection with P. capsici. When fruits
appeared healthy, three tissue sections were excised from under the point of inoculation
and plated onto BARF-amended UCV8. Plates were incubated at room temperature
under constant ﬂuorescent lighting for 3 to 5 days and observed using a compound
microscope. P. capsici was conﬁrmed using morphological characteristics according to
the Phytophthora spp. key by W aterhouse (52). The resulting P. capsici colonies were
transferred to fresh UCV8, and isolates were characterized for mating type and
mefenoxam sensitivity (26) to confirm they had the same phenotype as the inoculum.
Two independent experiments were conducted.

The temperature and relative humidity in each incubation chamber was monitored
for the duration of each experiment using WatchDog model 450 data loggers (Spectrum

Technologies, Plainﬁeld, IL). Data loggers were set to record temperature (°C) and

74

relative humidity (%) at 10 min intervals. The accuracy ratings of the data loggers were
i0.6°C and i3% relative humidity. Data were downloaded into a computer at the end of
each experiment. Since lighting was not continuous, slight photoperiodic ﬂuctuations in
temperature and relative humidity were observed.

Effects of relative humidity on disease development. Six incubation chambers
were placed into a growth chamber maintained at 25°C with ﬂuorescent lighting (16/8
day/night photoperiod and 95 mE of light intensity). Glycerol/water solutions (60, 70,
80. and 100% relative humidities) and a saturated salt solution (potassium acetate, 35%
relative humidity) were prepared as described in Forney and Brand] (10) and Winston
and Bates (22). One liter of solution was added to each incubation chamber to control the
relative humidity of that chamber (~35, 60, 70, 80, or 100%). Eight 2.5 cm diameter x 3
cm tall pieces of PVC pipe (stilts) were placed into the solution in the bottom of each
incubation chamber. A 29 cm x 20 cm screen made of two layers of hardware mesh (0.7
cm squares) was suspended above the glycerol or salt solution by the stilts. A piece of
clear tygon tubing was inserted into a 6.4-mm hole that had been drilled into the lid of the
incubation chamber and connected to a TopFin XP-l25 aquarium pump to circulate the
air within the chamber. The temperature and relative humidity (Table 3.1) in each
chamber were monitored as described above. For each relative humidity treatment,

twelve fruits were inoculated with a Single 10-ul droplet of zoospore suspension
containing ~2.5 x 10 zoospores. Six fru1ts were chosen arbitrarily and removed from

each incubation chamber at 3 and 4 dpi, and evaluated for disease as described above.
Six control cucumbers were treated as described above. Three independent experiments

were conducted for each isolate (SP98 and OP97).

75

Table 3.1. Average, maximum, and minimum relative humidities observed in incubation

chambers over the course of this study.

 

 

 

 

Actual RH
Expected

RHa Isolate SP98 Isolate OP97
Mean :E SEb Max,c Mind Mean i SE Max. Min.
35 32.0i0.2 33.0 20.7 35.7i0.2 47.5 20.7
60 59.3:E0.l 69.5 48.9 61.4i0.2 74.3 48.6
70 67.3i0.1 76.2 60.5 67.6i0.1 72.5 56.6
80 81.1i0.1 86.6 72.2 79.1i0.l 85.7 71.6
100 99110.0 100.0 86.6 97.5i0.1 100.0 89.8

 

a . . .

RH=relat1ve humidity (%)
b . . .

Mean i SE=mean relative humidity i standard error
e . . . .

Max.=max1mum relative humidity

d . . . . . .
Min.=minimum relative humidity

76

Effect of wounding on size-related resistance. For each cucumber size chosen,
20 surface disinfested fruits remained unwounded, and a shallow wound (~1-2 mm deep)

was created in the center of 20 fruits using a sterilized 1 mm dissecting needle. All of the

. . . . . . 4
fru1ts were inoculated Wlll'l a Single lO-ul droplet of water containing ~2.5 x 10

zoospores in the center of the fruit, over the wound, if present. Ten unwounded and 10
wounded cucumbers were inoculated with isolate SP98 and the remaining cucumbers
were inoculated with OP97. Inoculated fruits were incubated for 4 days at room
temperature in moist chambers under constant ﬂuorescent lighting and evaluated as
described above. Six control cucumbers were treated as described above. The
experiment was conducted three times.

Statistical analyses. All statistical analyses were performed using the SAS
statistical package version 9.1 (SAS Institute, Inc., Cary, NC). Data from 10 and 35°C
were removed prior to analysis, as no symptoms or signs of the disease were observed on
fruits incubated at these temperatures. Averages were calculated for the two
perpendicular measurements of lesion and pathogen growth diameters prior to statistical
analysis. A split plot design was used for the temperature studies with temperature as the
mainplot effect and time as the subplot effect. Isolate was the mainplot effect for the
relative humidity studies and relative humidity was the subplot effect. For the wounding
studies, treatment (fruit size x isolate) was the mainplot effect and wounding was the
subplot effect. Using the Proc Mixed procedure of SAS, data were subjected to split-plot
analysis of variance (ANOVA, P5005), and Fisher’s protected LSD was used for
separation of means (P5005) when effects were signiﬁcant. Prior to analyses, the

number of Sporangia produced in each lesion was log transformed for all experiments,

77

and the water-soaked lesion diameter was log transformed for the wounding experiments
to fulﬁll the assumption of normally distributed residuals.

The Proc LOESS procedure of SAS was used to perform loess nonparametric
regression analyses to model the relationship between temperature, incubation time, and
each of the response variables (water-soaked lesion diameter, pathogen growth diameter,
and sporangial production). The correlation between predicted and actual measures of
disease severity was used to measure goodness-of-ﬁt of the loess nonparameteric

regression model.

RESULTS

No symptoms or signs of disease were observed on cucumber fruits incubated at
10 or 35°C, and the pathogen was never recovered from fruits incubated at 35°C. P.
capsici was recovered from a small (<10%) number of cucumber fruits incubated at 10°C
(data not shown). Germination of at least some of the zoospores placed on these fruits
could be observed via stereomicroscopy. At 1 dpi, symptoms were not apparent on any
of the fruits, regardless of incubation temperature (Figure 3.1A), but P. capsici was
isolated from fruits, suggesting that infection had already occurred (data not shown).
Water-soaked lesions were apparent on fruits incubated at 20 to 30°C at 2 dpi (Figure
3.1B), but pathogen growth and sporulation were not visible. At 3 dpi, pathogen growth

and sporulation in lesions could be observed and measured (Figure 3.1C and D). Up to

2.7 x 106 sporangia were quantiﬁed from a single lesion at 4 dpi.

The interaction between temperature and time was Signiﬁcant for water-soaked

lesion diameter, pathogen growth diameter, and Sporangial production in the lesion

78

 

Figure 3.1. A, No symptoms of disease were observed on ﬁ'uits incubated at any
temperature (25°C shown) at 1 dpi (PI=point of inoculation). B, Water-soaking (WS) was
apparent on fruits incubated at 20 to 30°C at 2 dpi (30°C shown), but pathogen growth
outside of the point of inoculation (P1) was not observed. Signiﬁcant pathogen grth
and sporulation was apparent in water-soaked lesions on fruits incubated at 20 to 30°C at

3 (C & D) and 4 dpi.

79

(P<0.0001). Slicing of the interaction Showed differences in water-soaked lesion
diameter and pathogen growth diameter for the different incubation temperatures for
fruits incubated 3 or 4 dpi (P<0.0054), but not on fruit incubated for 1 or 2 dpi (P>0.22).
Differences in sporangial production in lesions were only observed at 4 dpi (P<0.0001).
The water-soaked lesion diameter was greatest for fruits incubated 4 dpi at 20 or 25°C.
At 3 dpi, the water-soaked lesion diameter was similar for fruits incubated at 20, 25, or
30°C (Figure 3.2A). The pathogen growth diameter and Sporangial production were
greatest for fruits incubated at 25°C at 4 dpi (Figure 3.2B, C). At 3 dpi, the diameter of
pathogen growth was greatest for fruits incubated at 30°C, and sporangial production was
similar for fruits incubated at 20, 25, or 30°C. The results of locss regression suggested
that 25°C was the most favorable temperature for disease development at 4 dpi (Figure
3.2E, F, G), in agreement with the results from ANOVA analyses. The main effect of
time was signiﬁcant (P<0.0001) for all of the response variables, but temperature was not
(P>0.07). Water-soaked lesion diameter, pathogen growth diameter, and sporangial
production were greatest at 4 dpi (Figure 3.2D).

No signiﬁcant interaction was noted between relative humidity, isolate, and time
(P>0.5731), but there was a signiﬁcant interaction between relative humidity and time for
all three measures of disease severity (P<0.043 7). The diameter of water-soaking and
pathogen growth increased as the relative humidity increased at both 3 and 4 dpi (Figure
3.3A, B). Sporangial production in the lesions was greater for cucumbers incubated at
260% (Figure 3.3) than for fruits incubated at 35% relative humidity at 3 or 4 dpi (Figure

3.3C). Sporangia were not produced (Figure 3.3C, E, F), but lesions did form (Figure

80

Figure 3.2. A, Mean water-soaked lesion diameter B, pathogen growth diameter and C,
number of sporangia produced on pickling cucumbers after 1, 2, 3, or 4 days incubation
at 15, 20, 25, or 30°C following inoculation with Phytophthora capsici zoospores. Note
differences in y axes and break in the y axis from 10-100 in C. Error bars represent
standard error. Bars that share letters are not signiﬁcantly different within a time point
(days post inoculation, dpi, lower case letters) or between time points (capital letters).
Response surface of the D, water-soaked lesion diameter, E, pathogen growth diameter
and F, number of Sporangia per lesion as a function of temperature and time (dpi). The
plots were generated using predicted values from ﬁtted loess nonparametric regression
models. Models were ﬁtted using 32 observations (two experiments x 16 treatment

combinations).

81

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82

3.3A, F) when inoculated fruits were incubated at 35% relative humidity. The interaction
between isolate and time was not Si gniﬁcant (P>0.2424), nor was the main effect of
isolate (P>0.08l3). The main effect of time was significant (P<0.0001) and greater
diameters of watersoaking and pathogen growth and sporangia production was observed
on fruits incubated for 4 dpi (Figure 3D). The interaction between isolate and relative
humidity was not signiﬁcant for the water-soaked or pathogen growth diameter
(P>0.0750), but it was signiﬁcant for sporangial production in the lesion (P=0.0059). For
isolate SP98, sporulation was significantly lower in lesions on fruits incubated at 35%
relative humidity when compared to the other relative humidites tested. For OP97,
sporulation was also lowest at 35%, but a statistically similar number of sporangia were
produced in lesions on cucumbers incubated at 60% relative humidity (Figure 3.3E).
When the main effect of relative humidity (P<0.0001) was examined, the water-soaked
lesion diameter was greater as the relative humidity level increased (Figure 3.3F).
Pathogen growth diameter followed a similar trend, except that the diameter for 70 and
80% relative humidites were Statistically similar. Sporangial production was greatest at
60% and lowest at 35% relative humidity, with a Statistically similar number of sporangia
observed in lesions on cucumbers incubated at the other relative humidity values tested
(Figure 3.3F).

The interaction between treatment (fruit Size x isolate) and wounding (wounded
or not) was Signiﬁcant for water-soaked lesion diameter (P<0.0001), pathogen growth
diameter (P<0.0001), and sporangial production in the lesion (P=0.0017). Small and
medium fruits inoculated with SP98 displayed the largest water-soaked lesion diameters

among the unwounded fruits. The smallest water-soaked lesions were observed on

83

Figure 3.3. Mean A, water-soaked lesion diameter B, pathogen growth diameter and C,
number of sporangia produced on pickling cucumber fruits after 3 or 4 days incubation at
35, 60, 70, 80, or 100% relative humidity following inoculation with Phytophthora
capsici zoospores. D, Mean water-soaked lesion diameter, pathogen growth diameter,
and number of sporangia produced on pickling cucumbers following 3 or 4 days
incubation. Bars that share letters are not signiﬁcantly different within a time point (days
post inoculation, dpi, lower case letters) or between time points (capital letters). E, The
number of sporangia produced on cucumbers incubated at 35, 60, 70, 80, or 100%
relative humidity by isolate. F, Mean water-soaked lesion diameter, pathogen growth
diameter, and number of sporangia produced on pickling cucumbers following incubation
under different conditions of relative humidity. Bars that share letters are not
signiﬁcantly different for a speciﬁc isolate (lower case letters) or between conditions of

relative humidity (capital letters). Error bars represent standard error.

84

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85

unwounded large fruits inoculated with isolate OP97 (Figure 3.4A). When fruits were
wounded. Similar water-soaked lesion diameters were observed for fruits of all Sizes, and
the diameter of water-soaking was greater than for unwounded fruits of the same Size and
isolate combination (Figure 3.4A). A similar relationship was also observed for pathogen
growth in the lesion (Figure 3.48) and sporulation (Figure 3.4C), with lesions on
wounded fruits Showing more pathogen growth and sporulation than on'corresponding
unwounded fruits. While pathogen growth was greater on wounded large fruits than on
unwounded large fruits, the pathogen growth diameter was still greater on unwounded
small and medium fruits (Figure 3.48). Pathogen growth diameter and sporulation were
greatest on wounded small or medium cucumbers inoculated with SP98 (Figure 3.4B
&C). Sporangia were not produced in lesions on large fruits, except for large wounded
fruits inoculated with SP98 (Figure 3.4C).

Overall, symptom expression was greater on fruits inoculated with SP98 than with
OP97 (Figure 3.4A) and the main effect of treatment (size x isolate) was signiﬁcant
(P<0.0012). All of the wounded fruits inoculated with SP98 had a greater water-soaked
lesion diameter than any of the unwounded fruits, regardless of isolate (Figure 3.4A). In
addition, wounded fruits inoculated with OP97 had lesions of similar size to the largest
lesions observed on the unwounded fruits (Figure 3.4A). Interestingly, the difference in
virulence observed between isolates on unwounded fruits was less obvious when the
fruits were wounded (Figure 3.4A). However, sporangial production was much greater
with SP98 than with OP97, regardless of whether or not the fruit were wounded (Figure
3.4C). Wounded fruits showed more severe symptoms and Signs of disease than

unwounded fruits (Figure 3.4) and the effect of wounding was signiﬁcant (P<0.0001).

86

Figure 3.4. Mean A, water-soaked lesion diameter, B, pathogen growth diameter and C,
number of Sporangia produced on pickling cucumber fruits of various sizes (small,
medium, or large) that were wounded or not 4 days post inoculation (dpi) with a droplet
of water containing Phytophthora capsici zoospores. Note differences in the y-axes.
Means that share letters are not Signiﬁcantly different for an isolate (lower case letters) or

between treatments (size*wounding. capital letters). Error bars represent standard error.

87

 

 

 

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88

Hence, wounding lessens Size-related. resistance.

DISCUSSION

This study showed that P. capsici is capable of infecting cucumber fruits under a
wide range of temperature and relative humidity conditions. In addition, wounding was
found to lessen size-related resistance to P. capsici in pickling cucumber. Careful
handling, storage, and transport of pickling cucumbers and temperatures <15°C are
recommended based on the results of this study. The current recommended storage
temperature of ~12°C (6) seems appropriate in light of these results.

Growth chamber studies showed that P. capsici is capable of infecting and
causing disease on cucumber fruits incubated at 15 to 30°C. By 2 dpi, water-soaked
lesions, the ﬁrst symptom of Phytophthora fruit rot, were present in this study, as has
been observed in previous studies in cucumber (15). A slightly longer lag has been
observed in pepper with symptom expression at 3 to 6 dpi (4). A limited number of
cucumbers were infected, but did not display symptoms when incubated at 10°C in this
study. The minimum growth temperature for P. capsici in culture medium has been
reported as 10°C (9), and it is not surprising that symptoms were not observed at this
temperature. Pickling cucumbers were infected in a previous study when incubated in a
large volume of zoospore suspension held at 9°C, removed from the suspension, and then
incubated at room temperature (13). A previous study in pepper (4) showed that disease
incidence was affected by the volume of P. capsici zoospore suspension used, even if the
same number of zoospores were present in the water. Since the volume of inoculum in

our studies was very small (10 pl), this may explain this discrepancy. In addition,

89

symptoms may have developed during incubation at room temperature. Cucumbers
incubated at 35°C did not Show symptoms of disease in this study, and the pathogen was
not recovered from these fruits, even though P. capsici has a growth maximum of >35°C
in culture medium (9,20,21). Both of the isolates used in this study grow readily on
UCV8 when incubated at 35°C (data not shown).

Disease development was signiﬁcant for cucumbers incubated at 20 to 30°C in
our study and limited lesions formed at 15°C. Symptoms and signs of disease were most
apparent on cucumbers incubated at 25°C for 4 dpi in our study, which was similar to
what was previously reported as ideal for infection and lesion development on pepper
fruits (27°C) (4) and leaves (23°C) (19) . The optimal growth temperature for P. capsici

isolates in culture media ranges from 24 to 33°C depending on the isolate tested (17). In

the pepper study fruits were not infected at 15 or 35°C when 5 x 1. 0J zoospores were

placed onto the fruits in a IOO-ul droplet of water (4), which was a lower concentration

than was used in our study (2.5 x 104 zoospores per lO-ul droplet of water). Sporangia

did not form in lesions on cucumber fruits in this study following exposure to
temperatures 5 15°C or 2 35°C, suggesting limited or no sporangial production at these
temperatures. AS would be expected, symptoms and Signs of disease became more
pronounced the longer the cucumbers were incubated.

Lesion expansion and pathogen growth were greater for cucumbers incubated at
high relative humidity. When fruits were incubated at 35% relative humidity, no
sporangia were detected in the lesions. However, P. capsici was able to produce
thousands of Sporangia in some of the lesions on fruits incubated at 260% relative

humidity. While Similar numbers of sporangia were produced at 260% at 3 dpi,

90

signiﬁcantly more sporangia were produced at 60% relative humidity than at 270% at 4
dpi in our study. Lamour and Hausbeck (15) found that sporangial production was
greater at 60% and 80% relative humidity than at >90% at 5 dpi on Slicing cucumber
fruits, in partial agreement with the current study. It is interesting to note that while 60%
was optimal for sporangial production by isolate SP98, this relationship was not as clear
for OP97. For OP97, sporulation was greater at 60 to 80% relative humidity and was
reduced at 100% relative humidity, in agreement with the results from Lamour and
Hausbeck (15) who also used isolate OP97 in a previous study.

In this study, all of the symptoms and signs of disease were greater on wounded
fruits than on unwounded fruits. Wounding has been Shown to increase P. capsici
disease severity in Fraser ﬁr (18), pepper plants (1), and pepper fruits (3) as well. In our
study, small and medium unwounded cucumbers showed more severe symptoms and
signs of disease than large fruits, as has been previously noted for pickling and slicing
cucumber (l2) and pepper (3) fruits. Gevens et a1. (12) found that oversized pickling
cucumbers had lower disease incidence, and no sporangia were detected in lesions on
pickling cucumbers 214 cm long. We did, not detect sporangia in lesions on unwounded
large fruits at 4 dpi, but sporangia were formed in the large fruits 214 cm long that were
wounded prior to inoculation. For isolate SP98, Sporangial production on wounded large
fruits was reduced compared to unwounded or wounded small or medium ﬁ'uits.
Sporangial production was generally poor on fruits inoculated with OP97 as compared to
SP98, but wounding increased sporangial production.

When cucumbers were not wounded, small and medium fruits had larger lesion

diameters than large fruits. However, wounding appeared to negate the difference in

91

lesion diameter; water-soaked lesion diameters were Similar for wounded fruits
inoculated with the same isolate regardless of fruit size. In pepper, wounding has been
found to negate age-related resistance and lesion expansion was similar on wounded
fruits, regardless of age (3). Interestingly, while differences in virulence by isolate were
obvious for unwounded fruits. differences in virulence on wounded cucumbers were
more subtle for water-soaked lesion diameter and pathogen growth diameter, suggesting
that virulence may be at least partially affected by the penetration ability of the different
pathogen isolates used. There were obvious differences between isolates for Sporangial
production on both unwounded and wounded fruits.

In summary, P. capsici is a dynamic pathogen that is capable of infecting and
causing disease on pickling cucumbers under a wide range of temperature and relative
humidity conditions. Because there is a lag time of days following inoculation before
symptom expression and sporulation, storing and transporting potentially infected fruits
under cool temperature (<15°C) is necessary to slow disease progression so that fruits
will not rot in transit. Wounding has been shown to lessen the effects of age-related
resistance; thus, careful handling of the fruits is key for cucumbers of all ages and Sizes.
Cucumber fruits may be dropped distances of >3 m during harvest and handling, which
constitutes a major source of mechanical injury for the fruits (32). If injured fruits are

exposed to inoculum after wounding, disease is more likely to occur.

92

ACKNOWLEDGMENTS
This research is based upon work supported by the USDA CSREES Special Research
Grant Awards 2009-34572-19990 and 2008-34572-19339, MAES multistate project NE-
1.035, and the Pickle and Pepper Research Committee of MSU, Pickle Packers
International, Inc. We thank A. Lebeis, M. Wood, and R. Heslip for technical assistance,
W. Wang for help with statistical analyses, and S. Linderman and L. Quesada—Ocampo

for critical review of the manuscript.

93

10.

ll.

12.

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95

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