FILTRATION OF PHYTOPHTHORA AND PYTHIUM ZOOSPORES
IN IRRIGATION WATER
By
Sangho Jeon

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Crop and Soil Sciences – Doctor of Philosophy
2017

ABSTRACT

FILTRATION OF PHYTOPHTHORA AND PYTHIUM ZOOSPORES
IN IRRIGATION WATER
By
Sangho Jeon
Phytophthora and Pythium are commonly known as water molds, and can cause
enormous damages to many floriculture and vegetable crops worldwide, including
seedling damping-off, stunting, and crown, stem and root rot. It is challenging to control
these pathogens because plants can be infected and do not show symptoms until the
disease is too advanced to respond to treatment. The pathogens can also easily develop
resistance to effective fungicides. As the zoospore movement with water flow is a major
transmission pathway of these pathogens, understanding the transport of zoospores in
natural and engineered systems is critical to developing strategies to control the
pathogens in both field and greenhouse crops. Thus, the first study investigated the
transport and retention of Phytophthora. capsici zoospores in saturated columns packed
with iron oxide coated sand (IOCS) or uncoated sand in Na+ or Ca2+ background solution
at pH 7.2 or 4.4, in combination with XDLVO interaction energy calculations and
microscopic visualizations. Significantly more encysted zoospores were retained in IOCS
than in uncoated sand, and at pH 4.4 than at pH 7.2, which likely resulted from increased
electrostatic attraction between zoospores and grain surface. At pH 7.2, up to 99% and 96%
of the encysted zoospores were removed in IOCS and uncoated sand, respectively, due to
a combination of strong surface attachment, pore straining, and adhesive interactions.
Motile biflagellate zoospores were more readily transported than encysted zoospores,
thus posing a greater dispersal and infection risk.

The second and third studies were conducted in a greenhouse to demonstrate a
proof-of-concept of using fast-flow filtration to control Phytophthora and Pythium
diseases in greenhouse floriculture and vegetable crops. The second study showed that
Pythium aphanidermatum could be effectively removed by the fast-flow sand and AC
filters at low water pressure. The rapid sand filter had the best performance because no
decrease in the poinsettia quality was observed when compared to the non-inoculated
control plants. Because the AC filter could also remove the essential nutrients from the
irrigation water, and cause the Fe deficiency in the poinsettias, it is less desirable to be
used unless the nutrients can be supplied separately instead of through irrigation water.
The third study found that the filter with iron oxide coated media [IOCM] could
effectively protect the squash plants from Phytophthora capsici, but caused the nutrient
deficiency in the squash. The sand filter could not prevent, but only slow the disease
development in the squash. Again, it shows that the IOCM filter has the potential to be
used in treating irrigation water in the greenhouse vegetable production, but sufficient
nutrients also need to be provided. Overall, the results suggested that physical removal of
pathogens using fast-flow filtration can overcome many limitations of fungicide
application, and may be a promising alternative for disease management in greenhouses.

This thesis is dedicated to my parents, Hyeongu Jeon and Oknam Choi, my wife
Yiseul Kim, and my daughter Yuna K. Jeon

iv

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Dr. Wei Zhang for his
guidance, encouragement, and motivation throughout my Ph.D. research. From him, I
have learned how to think critically toward scientific questions and problems. He always
shows me right directions whenever I face challenges. Thanks for showing me a fantastic
example of a scientist that I ever want to be. I would like to extend my thanks to all my
research committee members, Dr. Hui Li, Dr. Mary K. Hausbeck, and Dr. Steven
Safferman for their insightful comments and guidance.
My special thanks go to the former and present my group and Dr. Hausbeck’s lab
members especially, Charles Krasnow, Blair Harlan, Cheng-Hua Liu, Yike Shen, Jianzho
He, Dr. Yuan Tian, Gemini D. Bhalsod, Yingjie Zhang, Ya-Hui Chuang, Zeyou Chen,
and others. During the past years working with my group members, I learned not only
about experimental techniques but also generosity, decisiveness, and positive attitude as a
scientist.
I wish to thank my friends that I met in Michigan State University. Without their
support and encouragement in every way, I doubt about completion of my dissertation.
Adina Howe and Mike Howe provided unconditional support for the past years. They are
the most lovely and generous couple I have ever seen. I am thankful to Qingpeng Zhang
for entertaining me with the funniest stories and jokes. I will remember the wonderful
moments I shared with all of you.
Most importantly, I owe my deepest gratitude to my parents Hyeongu Jeon and
Oknam Choi and parents−in−law Youngseok Kim and Yeongae Kim. Thanks for their
v

unconditional love and support, and understanding for my decision to obtain my degree. I
wish to thank Yiesul Kim, who chose to spend her life with me and has supported me
with her tireless patience and tolerance toward my work and me for the past years. She is
an incredible mentor in my life who has shown me generosity and patience. She is an
excellent scientist, who always inspires me with creativity and enthusiasm for research.
She has truly made this possible. Special thanks go to my dear daughter Yuna K. Jeon.
She always shows me smile that makes me the happiest father in the world. Deep thanks
also go to my other family members my brother Byoungyong Jeon, sister-in-law Kum
Jeon, sister Jeongyeon Jeon, and brother-in-law Jongkeun Lee and Heonil Kim for their
unlimited love and support.

vi

TABLE OF CONTENTS

LIST OF TABLES ........................................................................................................... ix
LIST OF FIGURES ........................................................................................................ xii
CHAPTER 1 ...................................................................................................................... 1
1.1. INTRODUCTION ................................................................................................... 1
1.2. STUDY OBJECTIVES............................................................................................ 5
REFERENCES ............................................................................................................... 7
CHAPTER 2 .................................................................................................................... 10
TRANSPORT
AND
RETENTION
OF
PHYTOPHTHORA
CAPSICI
ZOOSPORES IN SATURATED POROUS MEDIA .................................................. 10
ABSTRACT.................................................................................................................. 10
2.1. INTRODUCTION ................................................................................................. 12
2.2. MATERIALS AND METHODS........................................................................... 15
2.2.1. Zoospore Suspensions ..................................................................................... 15
2.2.2. Porous Media .................................................................................................. 17
2.2.3. Column Experiments ...................................................................................... 17
2.2.4. XDLVO Calculations...................................................................................... 21
2.3. RESULTS AND DISCUSSION ............................................................................ 21
2.3.1. Characterization of Porous Media and Zoospores .......................................... 21
2.3.2. Retention of Encysted Zoospores ................................................................... 25
2.3.2.1. Effect of Iron Oxide Coating and Grain Size............................................... 25
2.3.2.2. Effect of Solution Chemistry ....................................................................... 26
2.3.2.3. Comparison of Biflagellate and Encysted Zoospores .................................. 32
2.4. ENVIRONMENTAL IMPLICATIONS................................................................ 34
2.5. ACKNOWLEDGMENTS ..................................................................................... 35
APPENDIX ................................................................................................................... 36
REFERENCES ............................................................................................................. 65
CHAPTER 3 .................................................................................................................... 73
FILTRATION OF PYTHIUM APHANIDERMATUM ZOOSPORES IN
RECYCLED IRRIGATION WATER TO CONTROL POINSETTIA DISEASE IN
GREENHOUSES ............................................................................................................ 73
ABSTRACT.................................................................................................................. 73
3.1. INTRODUCTION ................................................................................................. 74
3.2. MATERIALS AND METHODS........................................................................... 77
3.2.1. Irrigation and Filtration Systems. ................................................................... 77
3.2.2. Pathogen Culture and Inoculum...................................................................... 78
3.2.3. Plant and Irrigation Water. .............................................................................. 78
3.2.4. Greenhouse Experiments. ............................................................................... 79
vii

3.2.5. Plant Assessments. .......................................................................................... 81
3.2.6. Statistical Analysis. ......................................................................................... 82
3.3. RESULTS .............................................................................................................. 82
3.3.1. Irrigation and Filtration Systems. ................................................................... 82
3.3.2. Horticultural rating.......................................................................................... 84
3.3.3. Plant Assessment. ........................................................................................... 85
3.4. DISCUSSION ........................................................................................................ 93
3.4.1. Filtration Performance. ................................................................................... 93
3.4.2. Fungicide application. ..................................................................................... 95
3.4.3. Pathogen inter-plant transmission. .................................................................. 96
3.5. IMPLICATIONS ................................................................................................... 97
3.6. ACKNOWLEDGMENTS ..................................................................................... 98
APPENDIX ................................................................................................................... 99
REFERENCES ........................................................................................................... 127
CHAPTER 4 .................................................................................................................. 135
CONTROL OF PHYTOPHTHORA CAPSICI DISEASES IN GREENHOUSE
SQUASH BY FAST-FLOW FILTRATION .............................................................. 135
ABSTRACT................................................................................................................ 135
4.1. INTRODUCTION ............................................................................................... 136
4.2. MATERIALS AND METHODS......................................................................... 139
4.2.1. Irrigation and Filtration Systems. ................................................................. 139
4.2.2. Pathogen Culture and Inoculum.................................................................... 140
4.2.3. Greenhouse Experiments. ............................................................................. 141
4.2.4. Filtration Performance Assessments ............................................................. 142
4.2.5. Chemical Analyses........................................................................................ 143
4.2.6. Statistical Analysis ........................................................................................ 144
4.3. RESULTS ............................................................................................................ 144
4.3.1. Irrigation and Filtration Systems .................................................................. 144
4.3.2. Plant Assessment .......................................................................................... 146
4.4. DISCUSSION ...................................................................................................... 153
4.4.1. Filtration Performance .................................................................................. 153
4.4.2. Fungicide Application ................................................................................... 154
4.5. IMPLICATIONS ................................................................................................. 155
4.6. ACKNOWLEDGMENTS ................................................................................... 156
APPENDIX ................................................................................................................. 158
REFERENCES ........................................................................................................... 174
CHAPTER 5 .................................................................................................................. 181
CONCLUSION AND FUTURE WORK ................................................................... 181

viii

LIST OF TABLES

Table 1.1. Currently available disinfestation methods for recirculation systems and their
advantages, disadvantages, and characteristics. .......................................................... 6
Table 2.1. Characteristics of P. capsici Zoospore Suspensions and Their Transport
Parameters through Saturated Columns Packed with Iron Oxide Coated Sand (IOCS)
and Uncoated Sand.a ................................................................................................. 20
Table 2.S1. Electrophoretic Mobility (EPM) and Îś-potential of Uncoated and Iron Oxide
Coated Sand. ............................................................................................................. 44
Table 2.S2. XDLVO Calculations and Attachment Efficiency for an Encysted Zoospore
Interacting with the Solid-Water Interface (SWI).a .................................................. 49
Table 2.S3. Expt. 1: Biflagellate zoospores, IOCS, 500–804 µm, 0.11% Fe, pH 7.2 ± 0.2
................................................................................................................................... 51
Table 2.S4. Expt. 2: Encysted zoospores, IOCS, 500–804 µm, 0.11% Fe, pH 7.2 ± 0.2 . 52
Table 2.S5. Expt. 2: Encysted zoospores, IOCS, 500–804 µm, 0.11% Fe, pH 7.2 ± 0.2 . 53
Table 2.S6. Expt. 3: Encysted zoospores, Sand, 250~500 Âľm, 0% Fe, pH 7.2 Âą 0.2 ....... 54
Table 2.S7. Expt. 3: Encysted zoospores, Sand, 250~500 Âľm, 0% Fe, pH 7.2 Âą 0.2 ....... 55
Table 2.S 8. Expt. 4: Encysted zoospores, IOCS,250~500 Âľm, 0.13% Fe, pH 7.2 Âą 0.2 . 56
Table 2.S 9. Expt. 4: Encysted zoospores, IOCS,250~500 Âľm, 0.13% Fe, pH 7.2 Âą 0.2 . 57
Table 2.S 10. Expt. 5: Encysted zoospores, IOCS, 250~500 Âľm, 0.22% Fe, pH 7.2 ....... 58
Table 2.S 11. Expt. 6: Encysted zoospores, Sand, 500~804 Âľm, 0% Fe, pH 7.2 Âą 0.2 .... 59
Table 2.S 12. Expt. 6: Encysted zoospores, Sand, 500~804 Âľm, 0% Fe, pH 7.2 Âą 0.2 .... 60
Table 2.S 13. Expt. 7: Encysted zoospores, Sand, 250~500 Âľm, 0% Fe, pH 4.4 Âą 0.1 .... 61
Table 2.S 14. Tracer(NaBr), sand 250~500 µm, IS = 0, 𝐶0 = 100 mg L-1 ....................... 62
Table 2.S 15. Expt. 8: Encysted zoospores, IOCS, 250~500 Âľm, 0.13% Fe, pH 4.4 Âą 0.1
................................................................................................................................... 63

ix

Table 3.1. Root rot severity and horticultural rating in the first and second experiments. 88
Table 3.2. Chlorophyll a and b in the first and second experiments. ................................ 89
Table 3.S1. Results of nutrients in the irrigation water during the first and second
experiments. ............................................................................................................ 113
Table 3.S2. Micronutrients in the leaves of the poinsettias in the second experiment. .. 118
Table 3.S3. Experiment A: Poinsettia Foliar fresh weight (g) ........................................ 120
Table 3.S4. Experiment A: Poinsettia foliar dry weight (g) ........................................... 121
Table 3.S5. Experiment A: Poinsettia root dry weight (g) ............................................. 121
Table 3.S6. Experiment A: Poinsettia height (cm) ......................................................... 122
Table 3.S7. Experiment A: Poinsettia root rot severity .................................................. 122
Table 3.S8. Experiment A: Poinsettia chlorophyll a and b ............................................. 123
Table 3.S9. Experiment B: Poinsettia foliar fresh weight (g) ......................................... 124
Table 3.S10. Experiment B: Poinsettia foliar dry weight (g) ......................................... 124
Table 3.S11. Experiment B: Poinsettia height (cm) ....................................................... 125
Table 3.S12. Experiment B: Poinsettia root rot severity ................................................ 125
Table 3.S13. Experiment B: Poinsettia horticultural rank .............................................. 126
Table 3.S14. Experiment B: Poinsettia chlorophyll a and b (mg/ml) ............................. 126
Table 4.1. Percentage of the plants with wilting symptoms in the Experiment B. ......... 150
Table 4.S1. Nutrient concentrations in irrigation water during the Experiment B. ........ 165
Table 4.S2. Iron concentrations in the filtered and non-filtered irrigation water in the
Experiment A. ......................................................................................................... 166
Table 4.S3. Iron concentrations in the filtered and non-filtered irrigation water in the
Experiment B .......................................................................................................... 167
Table 4.S4. Zoospore concentrations in the Experiment A by colony forming units a. . 168
Table 4.S5. Experiment A: Squash foliar fresh weight (g) ............................................. 171
x

Table 4.S6. Experiment A: Squash foliar dry weight (g) ............................................... 171
Table 4.S7. Experiment A: Squash root fresh weight (g) ............................................... 172
Table 4.S8. Experiment A: Squash root dry weight (g) .................................................. 172
Table 4.S9. Experiment B: Squash foliar fresh weight (g) ............................................. 173
Table 4.S10. Experiment B: Squash foliar dry weight (g) .............................................. 173

xi

LIST OF FIGURES

Figure 2.1. Representative SEM images of encysted (A and C) and biflagellate (B) P.
capsici zoospores in CaCl2 solutions at pH 7.2 and IS 10 mM................................. 24
Figure 2.2. Breakthrough curves of encysted P. capsici zoospores in NaCl or CaCl2
solutions (pH 7.2 and IS 10 mM) through saturated columns packed with IOCS
(250−500 µm) of varying iron oxide coatings (A), IOCS and uncoated sand of
250−500 µm (B) and 500−804 µm (C). .................................................................... 29
Figure 2.3. Breakthrough curves of encysted P. capsici zoospores through saturated
columns packed with IOCS and uncoated sand at solution pH 4.4. ......................... 30
Figure 2.4. SEM images of encysted P. capsici zoospore attached on the IOCS surface in
the presence of Ca2+. ................................................................................................. 31
Figure 2.5. Breakthrough curves of biflagellate and encysted P. capsici zoospores through
saturated columns packed with IOCS (500–804 µm). .............................................. 33
Figure 2.S1. Observed P. capsici zoospore number concentrations in relation to
absorbance................................................................................................................. 38
Figure 2.S2. Schematic of preparation procedure for iron oxide coated sand (IOCS) (A),
and temporal visual changes of the preparation slurry (B) and IOCS (C). ............... 40
Figure 2.S3. Observed and fitted breakthrough curves of bromide tracer through saturated
column packed with uncoated sand (250−500 µm). ................................................. 41
Figure 2.S4. SEM images of Ottawa sand (A: 10,000 and C: 50,000) and iron oxide
coated sand (B: 10,000 and D: 50,000) of 250–500 µm. ................................... 42
Figure 2.S5. Energy-dispersive X-ray spectra of uncoated (A) and iron oxide coated sand
(B). ............................................................................................................................ 43
Figure 2.S6. SEM images of encysted (A, C and D) and biflagellate P. capsici zoospores
(B) in deionized water............................................................................................... 45
Figure 2.S7. SEM images of biflagellate (A) and encysted (B) P. capsici zoospores in
NaCl solution at pH 7.2 and ionic strength 10 mM. ................................................. 46
Figure 2.S8. SEM images of encysted P. capsici zoospores attached on iron oxide coated
surface in the presence of Na+. ................................................................................. 47

xii

Figure 2.S9. Total XDLVO interaction energies of zoospore interacting with uncoated
sand surface or iron oxide coated sand (IOCS) surface: (a) primary energy minimum
(ÎŚ1min) and maximum (ÎŚmax) and (b) secondary energy minimum (ÎŚ2min). ............. 64
Figure 3.1. Poinsettias at the end of the second experiment (i.e., 78 days after
inoculation). .............................................................................................................. 90
Figure 3.2. Roots of the poinsettias at the end of the first experiment (i.e., 69 days after
inoculation). IR: Infection ratio of Pythium in roots. ............................................... 91
Figure 3.3. Poinsettia height (A), foliar fresh biomass (B), foliar dry biomass (C), and
root dry biomass (D) in the first experiment (LSD test, P < 0.05). .......................... 92
Figure 3.S1. Schematic of the ebb-and-flow irrigation system constructed in the
greenhouse. ............................................................................................................. 103
Figure 3.S2. Schematic of filter unit (a. 12-V water pump, b. check valve, c. union fitting,
d: pressure sensor, e. PVC plug fitting, f. PVC adapter fitting, g. PVC coupling, h.
PVC end cap fitting, i. Bulkhead fitting, and j. motorized ball valve). .................. 104
Figure 3.S3. Location of pre-inoculated plants (red), infected plant (yellow), and healthy
plants (green) at the end of the first (A) and second (B) experiments. The number in
the column is the root rot severity. The infection of the plants was identified by the
isolation of Pythium aphanidermatum. ................................................................... 105
Figure 3.S4. Horticultural rating scale in the second experiment (#1 = high aesthetic
quality, and #5 = no aesthetic value). ..................................................................... 106
Figure 3.S5. Air temperature and relative humidity in the first (A) and second (B)
experiments. ............................................................................................................ 108
Figure 3.S6. Water pressure in the activated carbon (AC) and sand filters in the first (A)
and second (B) experiments. ................................................................................... 109
Figure 3.S7. pH of irrigation water measured in the first (A) and second (B) experiments.
................................................................................................................................. 110
Figure 3.S8. Electrical conductivity (EC) of irrigation water measured in the first (A) and
second (B) experiments........................................................................................... 111
Figure 3.S9. Irrigation water temperature in the holding tank for the first (A) and second
experiments (B). ...................................................................................................... 112
Figure 3.S10. Micronutrient concentrations in the irrigation water in the activated carbon
(AC) and non-inoculated control treatments during the second experiment. ......... 114

xiii

Figure 3.S11. Poinsettias at the end of the first experiment (i.e., 69 days after
inoculation). ............................................................................................................ 115
Figure 3.S12. Poinsettia roots for evaluating root necrosis in the first (A) and second (B)
experiments. White color of the roots indicates a healthy root system, and dark color
and dispersed potting soil suggest a rot root system. The number is the rating of root
rot severity (1 = no symptoms, 2 = mild root rot, <1/3 affected, 3 = intermediate root
rot, 1/3 to 2/3 affected, 4 = severe root rot, >2/3 roots affected, 5 = severe root and
crown rot, and 6 = dead plant). ............................................................................... 116
Figure 3.S13. Poinsettia height (A), foliar fresh biomass (B), and foliar dry biomass (C)
in the second experiment (LSD test, P < 0.05). ...................................................... 117
Figure 3.S14. Color of leaves comparison between non-inoculated control and AC filter
treatments in the first (A) and second (B) experiments. ......................................... 119
Figure 4.1. Images of squash plants at the end of the Experiment A (11 days after
inoculation). ............................................................................................................ 148
Figure 4.2. Representative images of squash plants at the end of the Experiment B (19
days after inoculation)............................................................................................. 149
Figure 4.3. Squash foliar fresh (A) and dry (B) weight, and root fresh (C) and dry (D)
weight in the Experiment A (LSD test, P < 0.05). .................................................. 151
Figure 4.4. Squash foliar fresh (A) and dry (B) weight in the Experiment B (LSD test, P <
0.05). ....................................................................................................................... 152
Figure 4.S1. Scheme of the ebb-and-flow irrigation system constructed in the greenhouse.
................................................................................................................................. 159
Figure 4.S2. Granular (A) and monolith (B) iron oxide coated media (IOCM). ............ 160
Figure 4.S3. Filter design of granular IOCM (A), sand (B), and monolith IOCM (C). . 161
Figure 4.S4. Air temperature and relative humidity during the Experiment A (A) and
Experiment B (B) in the greenhouse....................................................................... 162
Figure 4.S5. Water pressure in the sand filter in the Experiment A (A) and in the sand and
IOCM filters in the Experiment B (B). ................................................................... 163
Figure 4.S6. Water temperature in the holding tank during the Experiment A (A) and
Experiment B (B). ................................................................................................... 164
Figure 4.S7. Leaf and stem images of the squash plants in the Terrazole 35WP treatment
(A and B) and the non-inoculated control treatment (‒Control, C and D). ............ 169
xiv

Figure 4.S8. Root images at the end of the experiment A (i.e., 11 days after inoculation).
................................................................................................................................. 170

xv

CHAPTER 1

1.1. INTRODUCTION
Phytophthora and Pythium, also known as “water molds”, are a group of the most
notorious plant pathogens. Phytophthora and Pythium are fungus-like organisms that
cause seedling damping-off, stunting, and stem, crown, and root rot diseases in both
floriculture and vegetable crops, thus limiting their production and resulting in
devastating crop losses.1, 2 These pathogens are ubiquitous in the environment and infect
a wide range of greenhouse floral and vegetable crops. The water molds are challenging
to control because plants can be infected and do not show symptoms until the disease is
too developed to respond to any treatments.3 Moreover, they can easily adjust to the
environment especially under warm and wet conditions in a greenhouse. Thick-walled
oospores can survive for several years on plant containers, benches, floors, and in potting
media or soils. Furthermore, there are a limited number of fungicides effective against
these water molds, and pathogen resistance to effective fungicides is a primary concern.
These zoosporic organisms are generally found and well suited to the aquatic
environment for the movement and dissemination of their disease-inciting swimming
spores (i.e., zoospores).4 Water is the primary factor to release and transfer zoospores
from sporangia of Phytophthora and Pythium to a plant host. The disease epidemics are
mainly attributed to the rapid dispersal of the zoospores by flowing water during rainfall
and irrigation events, and easy recognition of a host tissue by either autotaxis,
chemotaxis, or electrotaxis. The movement of Phytophthora and Pythium zoospores in
the environment is dependent on their surface properties (including surface charges,
hydrophobicity, and bio-adhesive secretion), surface properties of porous media (e.g.,
1

surface charges, and grain size), as well as solution chemistry of the carrying water (e.g.,
solution pH, and ionic strength and composition). Understanding the zoospore movement
in porous media is not only important to the transport and retention of the zoospores in
soil profiles, but also to developing effective engineered filtration systems for the
pathogen removal.
In a greenhouse setting, the management of Phytophthora and Pythium is
particularly challenging, especially when irrigation water is recycled. Recycling irrigation
water including nutrients is beneficial both from environmental and economical
perspectives.4 Thus, irrigation water recycling in the greenhouses is becoming
increasingly popular. Meador et al.5 reported that 50% of the 24 surveyed greenhouses in
the U.S. recycled irrigation water, and the largest water users recycled 75–100% of their
irrigation water (490,000–1,000,000 gallons per day). However, this beneficial practice
can aggravate the transmission of water mold pathogens

6, 7

as their spread occurs when

the motile and encysted zoospores move with the flowing water. They may be introduced
to floriculture or vegetable crops in greenhouses via many ways including plant plugs or
other prefinished plant material. Or it can hibernate on dirty plant containers, benches,
and walkways or even ventilation systems. Hong and Moorman

4

reported that 16

Phytophthora spp. and 26 Pythium spp. were isolated from nursery and greenhouse
operations. Moreover, a survey conducted by Michigan State University (MSU) showed
that several Pythium spp. were identified from a wide range of floriculture crops.
Common Pythium spp. recovered from geranium were P. irregulare (68%) and P.
aphanidermatum (22%). Nearly all (96%) of the Pythium spp. recovered from
snapdragon were P. irregulare. Pythium spp. isolated from hibiscus included P.

2

irregulare (50%) and P. segnitium (50%). P. irregulare infects most lantana (83%). The
most prevalent Pythium spp. isolated from poinsettia included P. ultimum (53%) and P.
aphanidermatum. More importantly, the cycle of plant infection and zoospore production
is very short through asexual reproduction.8, 9 Therefore, even low levels of Phytophthora
and Pythium in recycled irrigation water can result in a rapid epidemic if the irrigation
water is not properly treated.
Various chemical and physical methods have been proposed to disinfect the
contaminated irrigation water in greenhouses, including filtration, chlorination, copper
ionization, ozonation, UV light, activated peroxygens, chlorine dioxide, and heat.4,

10

Advantages, disadvantages, and characteristics of these methods are summarized in Table
1.1. Chemical treatments are known to introduce resistance among pathogen populations,
which eventually make these treatments less effective.11 Moreover, a high level of
chemical dose may cause phytotoxicity to crops.12 Therefore, filtration is an appealing
cost-effective treatment to remove Pythium and Phytophthora from contaminated water.4,
13, 14

Currently, screen or disk filters with a pore size > 100 Îźm are used to remove large

particles such as potting soil particles and plant debris to avoid clogging of the drippers.5,
10

Also, these filters with large pore size are used as pre-treatment to improve the

efficiency of other disinfection methods such as heat treatment, ozone treatment, or UV
radiation.10 Membrane filters of relatively small pore size (<5Âľm, micro-filtration) can be
used to remove the water mold pathogens, but requires relatively high water pressure and
high maintenance cost due to clogging and leaking problems.15 Slow sand filtration has
been used for drinking water treatment, and also investigated for the removal of water
molds in the greenhouse applications.13, 16-18 However, it has not been used in commercial

3

greenhouse.12 Slow sand filters are easy to operate and maintain. Water flows slowly
through a packed bed of granular sand media at a rate of 100–300 L/m2/h.12, 15 However,
the water flow is too low to meet the water demand in a typical greenhouse, which
severely limits the wide adoption of filtration technique. Additionally, variable
performance has been observed with sand filtration systems.14,

19

Clearly, additional

research on fast-flow filtration is needed to first show the proof-of-concept of using
filtration systems in controlling the plant pathogens in the greenhouses. Better design of
filtration systems is often dependent on fundamental mechanisms controlling the
pathogen transport and retention in porous media.

4

1.2. STUDY OBJECTIVES
In light of the above overview, the objectives of this work were to the following
I. Investigate the transport and retention of Phytophthora capsici zoospores in
saturated sand columns as influenced by environmental factors such as sand grain size,
iron oxide grain coating, solution pH, and cation type. The mobility of biflagellate and
encysted zoospores was also compared to elucidate the role of encystment in the
zoospore transport.
II. Investigate the effectiveness of fast-flow filtration to control Pythium root rot
in potted poinsettias in greenhouses using the ebb-and-flow and flood-floor irrigation
systems. Two greenhouse experiments were performed to investigate the effect of filter
media type (i.e., sand and activated carbon), fungicide application (i.e, etridiazole), and
pathogen transmission mode (i.e., inoculation in plants vs in irrigation water).
III. Investigate the effectiveness of fast-flow filtration to control Phytophthora
diseases for potted squashes in greenhouses using the ebb-and-flow and flood-floor
irrigation systems. Two greenhouse experiments were carried out to test the effect of
filter media type (i.e., sand and iron oxide coated media [IOCM]) and fungicide
application (i.e, etridiazole).
The following chapters address the three objectives of my research. The Objective
I is addressed in Chapter 2, Objective II in Chapter 3, and Objective III in Chapter 4. The
dissertation ends with Chapter 5 that summarizes the findings of the studies that have
been conducted and identifies future research directions.

5

Table 1.1. Currently available disinfestation methods for recirculation systems and their advantages, disadvantages, and
characteristics.
Method of disinfestation

Advantages

Disadvantages

Characteristics

Non-Chemical Methods
1. Heat treatment

Highly effective

very energy intensive

temperature setpoint (95°C)
exposure time 10s
Wavelengths between 200 and 280 nm

2.

UV Radiation

Low space requirement

Efficiency drops with high organic matter and
bulb age
Interaction with micronutrients

3.

Filtration
a. Screen filtration

Low cost

No pathogen removal

Remove large particles
Pre-treatment for heat, ozone, and UV
radiation

b.

Membrane filtration

Highly effective

Size based filtration

c.

Slow sand filtration

Low cost

Frequent plugging and leaks
High capital costs
High space requirement
Effectiveness varies with pathogen
Low flow rate

Highly effective

High capital costs
High maintenance costs
Efficiency drops with high organic matter
Reacts with iron chelate

Not popular because of harmful for human
and strict rules

Low cost

not efficient

Different dosages are recommended

Low cost

Phytotoxicity at high concentrations

Strong oxidation including Sodium
hypochlorite(NaOCl),
Chlorine Dioxide (ClO2), and sodium
hypochlorite

Low cost

Phytotoxicity at high concentrations
Few fungicides available for water mold
pathogen
Pathogen quickly develop residence to the
effective fungicides

Chemical methods
1. Ozone (O3)

2.
3.

Hydrogen
(H2O2)
Chlorine

4.

Fungicide

peroxide

6

Bio-film

REFERENCES

7

REFERENCES

1.

Granke, L. L.; Quesada-Ocampo, L.; Lamour, K.; Hausbeck, M. K., Advances in
Research on Phytophthora capsici on Vegetable Crops in The United States. Plant
Dis 2012, 96, (11), 1588-1600.

2.

Themann, K.; Werres, S.; Luttmann, R.; Diener, H. A., Observations of
Phytophthora spp. in water recirculation systems in commercial hardy ornamental
nursery stock. Eur J Plant Pathol 2002, 108, (4), 337-343.

3.

Olson, H. A.; Benson, D. M., Characterization of Phytophthora spp. on Floriculture
Crops in North Carolina. Plant Dis 2011, 95, (8), 1013-1020.

4.

Hong, C. X.; Moorman, G. W., Plant pathogens in irrigation water: Challenges and
opportunities. Crit. Rev. Plant Sci. 2005, 24, (3), 189-208.

5.

Meador et al., Survey of physical, chemical, and microbial water quality in
greenhouse and nursery irrigation water. HortTechnology 2012, 22(6), 778-786.

6.

Hong, C. X.; Richardson, P. A.; Kong, P.; Bush, E. A., Efficacy of chlorine on
multiple species of Phytophthora in recycled nursery irrigation water. Plant Dis
2003, 87, (10), 1183-1189.

7.

Stanghellini, M. E.; Rasmussen, S. L., Identification and Origin of Plant-Pathogenic
Microorganisms in Recirculating Nutrient Solutions. Adv Space Res 1994, 14, (11),
349-355.

8.

Gevens, A.; Donahoo, R.; Lamour, K.; Hausbeck, M., Characterization of
Phytophthora capsici from Michigan surface irrigation water. Phytopathology
2007, 97, (4), 421-428.

9.

Erwin, D. C.; Ribeiro, O. K., Phytophthora diseases worldwide. American
Phytopathological Society (APS Press): 1996.

10.

van Os, E. A., Comparison of Some Chemical and Non-Chemical Treatments to
Disinfect a Recirculating Nutrient Solution. International Symposium on Soilless
Culture and Hydroponics 2009, 843, 229-234.

11.

Lamour, K. H.; Daughtrey, M. L.; Benson, D. M.; Hwang, J.; Hausbeck, M. K.,
Etiology of Phytophthora drechsleri and P. nicotianae (=P. parasitica) diseases
affecting floriculture crops. Plant Dis. 2003, 87, (7), 854-858.

12.

Raudales, R. E.; Parke, J. L.; Guy, C. L.; Fisher, P. R., Control of waterborne
microbes in irrigation: A review. Agr Water Manage 2014, 143, 9-28.

8

13.

Calvo-Bado, L. A.; Pettitt, T. R.; Parsons, N.; Petch, G. M.; Morgan, J. A. W.;
Whipps, J. M., Spatial and temporal analysis of the microbial community in slow
sand filters used for treating horticultural irrigation water. Applied and
environmental microbiology 2003, 69, (4), 2116-2125.

14.

MartĂ­nez, F.; Castillo, S.; Carmona, E.; AvilĂŠs, M., Dissemination of Phytophthora
cactorum, cause of crown rot in strawberry, in open and closed soilless growing
systems and the potential for control using slow sand filtration. Scientia
horticulturae 2010, 125, (4), 756-760.

15.

Ehret, D. L.; Alsanius, B.; Wohanka, W.; Menzies, J. G.; Utkhede, R.,
Disinfestation of recirculating nutrient solutions in greenhouse horticulture.
Agronomie 2001, 21, (4), 323-339.

16.

Lee, E.; Oki, L. R., Slow sand filters effectively reduce Phytophthora after a
pathogen switch from Fusarium and a simulated pump failure. Water Res 2013, 47,
(14), 5121-5129.

17.

Ufer, T., Werres, S. K., Posner, M., and Wessels, H.-P., Filtration to eliminate
Phytoph-thora spp. from recirculating water systems in commercial nurseries.
Online Plant Health Progress 2008, http://dx.doi.org/10.1094/PHP-2008-0314-01RS.

18.

van Os, E. A.; Amsing, J. J.; van Kuik, A. J.; Willers, H., Slow sand filtration: A
potential method for the elimination of pathogens and nematodes in recirculating
nutrient solutions from glasshouse-grown crops. International Symposium on
Growing Media and Hydroponics, Vols I and Ii 1998, (481), 519-526.

19.

DĂŠniel, F.; Rey, P.; ChĂŠrif, M.; Guillou, A.; Tirilly, Y., Indigenous bacteria with
antagonistic and plant-growth-promoting activities improve slow-filtration
efficiency in soilless cultivation. Canadian journal of microbiology 2004, 50, (7),
499-508.

9

CHAPTER 2

TRANSPORT AND RETENTION OF PHYTOPHTHORA CAPSICI
ZOOSPORES IN SATURATED POROUS MEDIA
This chapter has been published in Jeon, Sangho, Charles S. Krasnow, Caitlin K. Kirby,
Leah L. Granke, Mary K. Hausbeck, and Wei Zhang. "Transport and Retention of
Phytophthora capsici Zoospores in Saturated Porous Media."Environmental science &
technology”, no. 17 (2016): 9270-9278.

ABSTRACT
Phytophthora capsici is an important plant pathogen capable of infecting several
major vegetable crops. Water-induced P. capsici transport is considered to be a
significant contributor to disease outbreaks and subsequent crop loss. However, little is
known about factors controlling P. capsici zoospore transport in porous media, thus
impeding my understanding of their environmental dispersal and development of
filtration techniques for contaminated irrigation water. This study investigated the
transport and retention of P. capsici zoospores in saturated columns packed with iron
oxide coated sand (IOCS) or uncoated sand in Na+ or Ca2+ background solution at pH 7.2
or 4.4, in combination with XDLVO interaction energy calculations and microscopic
visualizations. Significantly more encysted zoospores were retained in IOCS than in
uncoated sand, and at pH 4.4 than at pH 7.2, which likely resulted from increased
electrostatic attraction between zoospores and grain surface. At pH 7.2, up to 99% and 96%
of the encysted zoospores were removed in IOCS and uncoated sand, respectively, due to
a combination of strong surface attachment, pore straining, and adhesive interactions.
Motile biflagellate zoospores were more readily transported than encysted zoospores,
thus posing a greater dispersal and infection risk. This study has broad implications in

10

environmental transport of Phytophthora zoospores in natural soils as well as in costeffective engineered filtration systems.

11

2.1. INTRODUCTION
Plant pathogens cause annual crop loss of approximately $33 billion in the US
despite large pesticide use for disease control; subsequent environmental release of used
pesticides can also lead to enormous human and ecosystem health costs.20, 21 Control of
plant pathogens, specifically those that can be disseminated through water flow, requires
a better understanding of their transport in porous media such as soils. However, studies
on the transport of plant pathogens (e.g., Phytophthora) in porous media are surprisingly
scarce.22 This is in contrast to extensive work regarding the transport of microbial human
pathogens (e.g., bacteria, virus, and protozoa) for the purpose of protecting water quality
and human health.23-26 This paucity of research impedes the development of effective
measures to mitigate environmental dispersal of plant pathogens. In the era of “one
health”, the research on plant pathogen transport is equally important in the context of
food security and protection of human and environmental health.
In this study I primarily focused on the oomycete Phytophthora capsici, a fungallike organism that infects many vegetables in the field and greenhouse.1, 4, 27, 28 Yield loss
from P. capsici infection was reported to be 40–100% for peppers, 20% for squash, and
65% for greenhouse-grown cucumbers.28 Sometimes total crop loss can occur due to P.
capsici outbreaks.27 Each year in Michigan alone, up to 25% of the state’s $134 million
vegetable value may be lost due to diseases caused by P. capsici.27 Hence, control of P.
capsici outbreaks is critical to many agricultural producers, and requires an integrated
management approach that targets pathogen survival, development and dispersal.1, 27, 29
Among recommended mitigation strategies for P. capsici (e.g., crop rotation, irrigation
management, fungicide treatment, and host-plant resistance), soil water management to

12

minimize water-induced pathogen dissemination within a field or greenhouse is important
to the successful control of this pathogen.1, 27, 29 A key feature of the P. capsici disease
cycle is the production of 20–40 biflagellate swimming spores (i.e., zoospores) from a
single non-motile sporangium on infected plant tissue upon exposure to rainfall, irrigation
water, or surface runoff.1, 27 Each of thousands of released zoospores has the potential to
infect a host plant. Thus, the transport of virulent P. capsici zoospores promotes plant
disease development.1, 8, 28 The flow-induced pathogen transmission in soils spreads the
pathogen across fields, and the pathogen-laden drainage water could contaminate shallow
groundwater or surface water. Irrigation with surface water contaminated with P. capsici
is an important contributor to disease outbreaks in field-grown vegetable crops.1,

8, 28

When contaminated surface water cannot be avoided, treating the water with rapid sand
filtration and chlorination has been implemented in the field.30
Similarly, in greenhouses disease outbreaks are often exacerbated by recycling
irrigation water.4, 31, 32 Irrigation water recycling is widely practiced by many greenhouse
growers to reduce pollutant discharge or alleviate water shortage.32, 33 For instance, some
greenhouses with significant water use (i.e., 0.5–1 million gallons a day) recycle 75–
100% of their irrigation water.34 Treatment of recycled irrigation water containing
pathogens is common in greenhouses and includes physical, chemical and ecological
methods.4, 35, 36 Plants are often treated with fungicides to reduce disease, but fungicides
are not always efficacious. Sustained fungicide treatment may prompt the development of
fungicide resistance within P. capsici populations.37,

38

High chemical doses can also

cause phytotoxicity to some crops 35 and pose significant environmental risk. In contrast,
physical removal by filtration does not have the abovementioned limitations, and can be

13

an attractive alternative to control the pathogen transmission in recirculating systems.4, 13,
14, 31, 39

Nonetheless, filtration techniques still need to be refined to improve performance

and facilitate adoption.36
Hence, control of P. capsici dispersal in the field and greenhouse requires
mechanistic understanding of factors governing the transport of zoospores in porous
media. A limited number of studies revealed that water-induced passive movement of
Phytophthora zoospores is more effective in transporting them over a greater distance in
soils than the autonomous, active movement of zoospore swimming.40,

41

Active

movement only allows the zoospores to move a maximum of a few millimeters to
centimeters,41-43 and collision with solid surface often results in encystment and a
subsequent loss in motility.41,

44, 45

However, the studies on the passive transport of

Phytophthora zoospores have been very limited.22 Transport of Phytophthora zoospores
in saturated porous media may be determined by several well-known colloid retention
mechanisms, including attachment at the solid-water interface, straining at the grain-tograin contacts, and mechanical filtration due to size-constraint at pore throats.23
Nonetheless, Phytophthora zoospores differ from many previously studied biocolloids
(e.g., bacteria, virus and protozoa) due to their relatively large size (~ 7.5 Âľm) and its
encystment phase. Encystment could even occur in bulk solution without surface
collision. During encystment, zoospores lose their flagella and are transformed from oval
shape to spherical shape.46, 47 Size and shape of colloids significantly affect their transport
behavior,

48-51

in addition to many environmental factors such as solution chemistry,

collector grain size, and surface properties of colloids and collector grains.23 Therefore, in
order to fill the knowledge gap on plant pathogen transport, this study aimed to

14

investigate the transport and retention of P. capsici zoospores in saturated sand columns
as influenced by environmental factors including grain size, iron oxide grain coating,
solution pH, and cation type. The mobility of biflagellate and encysted zoospores was
also compared to elucidate the role of encystment in the zoospore transport. This study
combined column transport experiments, microscopic visualization, and surface
interaction energy calculations to elucidate retention mechanisms of P. capsici zoospores
in porous media.

2.2. MATERIALS AND METHODS
2.2.1. Zoospore Suspensions
Phytophthora capsici isolate 11127 used in this study was obtained from the
culture collection maintained in the laboratory of M.K. Hausbeck at Michigan State
University. Isolate 11127 was originally isolated from a wax bean leaf from a field in
Van Buren County near Keeler, MI and was characterized as an A1 mating type and
insensitive to the fungicide mefenoxam.52 This isolate was selected because it could
produce a large number of zoospores needed for column transport experiments. Detailed
procedures for producing zoospores is provided in Supporting Information S1. The
prepared motile biflagellate zoospore suspensions were hand-shaken vigorously for 90
seconds to induce encystment; shaking was omitted for the motile zoospore suspensions
that were used immediately in subsequent column experiments. The zoospore number in
the stock suspensions was determined with a hemacytometer and then diluted to obtain
the testing zoospore suspensions under desired solution chemistry. The absorbance of the
testing suspensions was measured to be 0.174 Âą 0.024 at 600 nm by a UV-Visible

15

Spectrophotometer (Varian Cary 50 Bio, McKinley, New York). The zoospore
concentration in the testing suspensions was approximately 4.4 ± 2.2 × 105 zoospores
mL−1. There was a linear relationship between the zoospore concentration and the
absorbance (r2 = 0.999, Figure 2.S1). During dilution, background solution chemistry of
the testing zoospore suspensions was adjusted to 0.4 mM NaHCO3 + 9.6 mM NaCl and
0.4 mM NaHCO3 + 3.2 mM CaCl2 (pH = 7.2 Âą 0.2, ionic strength [IS] = 10 mM),
respectively. In order to investigate the effect of solution pH on the zoospore transport,
the testing zoospore suspensions at pH 4.4 Âą 0.1 were similarly prepared by adjusting
solution chemistry to 0.4 mM CH3COONa + 2.6 mM CH3COOH + 9.6 mM NaCl and 0.4
mM CH3COONa + 2.6 mM CH3COOH + 3.2 mM CaCl2 (pH 4.4 Âą 0.1, IS 10 mM),
respectively. The testing suspensions of motile biflagellate zoospores were prepared at
solution pH 7.2 to compare their mobility with that of the encysted zoospores.
Îś-potentials of the zoospore suspensions were measured using a Malvern
ZetaSizer Nano ZS (Malvern, Westborough, MA) that employs phase analysis light
scattering (PALS) to measure electrophoretic mobility (EPM) of charged particles. The
Smoluchowski equation was used to calculate the Îś-potentials from the EPM values.
Additionally, the zoospores were fixed by a chemical procedure and imaged by scanning
electron microscopy (SEM) as detailed in Supporting Information S1. The SEM images
were then analyzed by ImageJ 1.48v (Wayne Rasband, National Institutes of Health,
USA) using elliptical and spherical models for the biflagellate and encysted zoospores,
respectively. The length of major and minor axes were measured for the biflagellate
zoospores, whereas the diameter was measured for the encysted zoospores.

16

2.2.2. Porous Media
Ottawa sand (99.69% silica, GranusilÂŽ ) was obtained from Unimin Corporation
(Le Seueur, MN). The sand was sieved into fractions of 250–500 m and 500–804 m,
washed thoroughly as per Bradford et al.,53 dried, and stored in a closed glass bottle. Iron
oxide coated sand (IOCS) was prepared following the procedure modified from
Schwertmann and Cornell54 as described in Supporting Information 2.S1 (Figure 2.S2).
Two IOCS with lower and higher iron content (0.11–0.13% and 0.22% by mass) were
prepared. The sand grains were mounted on aluminum stubs with carbon cement (SPI
supplies, West Chester, PA) to observe the surfaces of uncoated sand and IOCS by SEM.
Similar to the previous approach,55, 56 the suspensions of IOCS (0.13% Fe) and uncoated
sand colloids were generated as described in Supporting Information S1 for the
measurements of Îś-potential by the zetasizer.
2.2.3. Column Experiments
Eight column transport experiment sets (Expt. 1.8) were performed to examine
the effects of zoospore type (i.e., biflagellate vs encysted zoospores), solution chemistry
(i.e., pH and ionic composition), and sand grain properties (i.e., iron oxide coating and
grain size) on the mobility of P. capsici zoospores in saturated porous media, as shown in
Table 2.1. In each experiment, the NaCl and CaCl2 treatments were conducted in parallel
to investigate the effect of cation type in background solution on the zoospore mobility.
More specifically, as shown in Table 2.1, Exp. 1 and Exp. 2 compared the difference
between the mobility of biflagellate and encysted zoospores. Exp. 3, 4 and 5 and Exp. 2
and 6 examined the effect of iron oxide coating and coated iron content on the transport
of encysted zoospores in two sand size fractions (i.e., 250–500 µm and 500–804 µm),

17

respectively. Exp. 2 and 4 and Exp. 3 and 6 explored the effect of grain size on the
transport of encysted zoospores in the presence and absence of iron oxide surface
coatings. Finally, Exp. 7 and 8 examined the transport of encysted zoospores at solution
pH 4, which was compared with their transport at solution pH 7.2 (i.e., Exp. 3 and 4 in
Table 2.1).
A glass column of 12.4.cm in length and 1-cm in inner diameter (Omnifit, Diba
Industries, Danbury, CT) was wet-packed with either the uncoated sand or IOCS to a
porosity of 0.35. The packed porous media were supported by stainless steel mesh of 104
Âľm opening size and sealed with O-rings. Two separate columns were used for the
zoospore suspensions in 3.2 mM CaCl2 or 9.6 mM NaCl background solution buffered
with 0.4 mM NaHCO3 (i.e., pH = 7.2 Âą 0.2 and IS = 10 mM), which were run at the same
time. The zoospore-free background solution was pumped through the column using a
syringe pump (Model 351, Sage Instruments, White Plains, NY) at about 0.2 mL min−1
for 45 minutes to equilibrate the column. The testing zoospore suspension of 3.1 ± 2.0 ×
105 zoospores mL−1 or a solution of bromide tracer at 50 mg L−1 was injected for 30
minutes at the identical flow rate, followed by injecting the background solution for 90
minutes. An infusion and withdrawal syringe pump (Model 74905-54, Cole-Parmer, IL)
was used in the bromide tracer experiment. The average pore water velocity was 0.77 Âą
0.04 cm min−1. The effluent samples were collected in 5-minutes interval using a fraction
collector (Retriever 500, Teledyne ISCO) and measured for absorbance at 600 nm for the
zoospore effluent samples and at 204 nm for the bromide effluent samples using the UVvisible spectrophotometer. The background absorbance (0.0004 Âą 0.0005) of the effluents
was subtracted from the effluent sample absorbance. The breakthrough curves were

18

plotted as normalized effluent concentrations as a function of pore volumes. Effluent
mass recovery (MER) was calculated by integrating the BTCs using the trapezoidal rule
and then dividing the recovered mass by the input mass. The transport of zoospores or the
conservative Br tracer is described by a convection-dispersion equation with a first-order
kinetic deposition term.

C
 2C
C
 D 2 v
 kd C
t
z
z

(1)

where C is the liquid phase concentrations, t is the elapsed time, D is the hydrodynamic
dispersion coefficient, 𝑧 is the travel distance, 𝑣 is the pore water velocity, and kd is the
deposition rate coefficient. For the bromide tracer kd is equal to zero. Thus, the bromide
BTC was fitted to the convection-dispersion equation to characterize hydrodynamic
properties of the column (Figure 2.S3). The estimated D value was 0.055 cm2 min−1, and
the flow regime in the columns was convection-dominant, characterized by a high PĂŠclet
number (Pe = vL/D = 161, where L is the column length).57, 58 Therefore, the dispersion
term in Eq. 1 can be ignored, and kd was estimated as follows:57, 59, 60

kd  

v
ln( M ER )
L

(2)

At the end of column experiments, the zoospores attached on IOCS were taken
out from the column using spatula, and observed with SEM. Briefly, the samples were
fixed at 4 °C for 12 hours in a 4% glutaraldehyde solution buffered with 0.1M sodium
phosphate at pH 7.4. Following a brief rinse in the buffer, samples were dehydrated in an
ethanol series (25%, 40%, 50%, 60%, 75%, and 95%) for 30 minutes at each gradation
and with three 30-min changes in 100% ethanol. The remaining procedure was the same
as that for the zoospore SEM imaging.
19

Table 2.1. Characteristics of P. capsici Zoospore Suspensions and Their Transport Parameters through Saturated Columns
Packed with Iron Oxide Coated Sand (IOCS) and Uncoated Sand.a
Experiment

Zoospore

(n)

type

Exp. 1

Biflagellate

(n =2)

zoospores

Exp. 2

Media Type

Solution

Cation

EPM

pH

Type

(µm cm V−1s−1 )

Na+

Îś-potential (mV)

MER

kd (min−1)

−1.41 ± 0.11 cd

−18.0 ± 1.4 cd

0.139 Âą 0.015 a

0.122 Âą 0.011 f

IOCS, 500–804

Ca

−0.92 ± 0.06 ab

−11.8 ± 0.7 ab

0.018 Âą 0.013 de

0.260 Âą 0.043 abc

Âľm, 0.11% Fe

Na+

−1.30 ± 0.17 c

−16.6 ± 2.2 c

0.030 Âą 0.009 cd

0.216 Âą 0.023 cde

Ca2+

−0.75 ± 0.05 a

−9.5 ± 0.7 a

0.014 Âą 0.002 de

0.266 Âą 0.016 ab

Na+

−1.43 ± 0.08 cd

−18.2 ± 0.8 cd

0.059 Âą 0.008 b

0.174 Âą 0.004 ef

Ca2+

−1.09 ± 0.20 b

−13.9 ± 2.5 b

0.058 Âą 0.016 b

0.168 Âą 0.017 ef

2+

(n =3)
Exp. 3

Sand, 250~500

(n =3)

Âľm, 0% Fe
7.2 Âą 0.2

Exp. 4

IOCS,250~500

Na

−1.55 ± 0.11 d

−19.8 ± 1.4 d

0.026 Âą 0.012 cde

0.227 Âą 0.036 bcd

(n =3)

Âľm, 0.13% Fe

Ca2+

−1.06 ± 0.06 b

−13.5 ± 0.8 b

0.023 Âą 0.008 cde

0.241 Âą 0.035 bcd

+

Exp. 5

Encysted

IOCS, 250~500

Na

−1.43 ± 0.04 cd

−18.3 ± 0.5 cd

0.011 Âą 0.003 de

0.312 Âą 0.011 a

(n =2)

zoospores

Âľm, 0.22% Fe

Ca2+

−1.05 ± 0.25 b

−13.4 ± 3.3 b

0.007 Âą 0.006 e

0.310 Âą 0.046 a

+

Exp. 6

Sand, 500~804

Na

−1.33 ± 0.06 c

−16.9 ± 0.8 c

0.042 Âą 0.017 bc

0.208 Âą 0.038 cde

(n =3)

Âľm, 0% Fe

Ca2+

−0.81 ± 0.09 a

−10.3 ± 1.1 a

0.042 Âą 0.016 bc

0.205 Âą 0.027 de

Exp. 7

Sand, 250~500

Na+

−0.77

−9.9

0.019

0.243

Ca

−0.61

−7.9

0.007

0.314

Na+

−0.78

−9.9

0.004

0.334

−0.60

−7.7

0.002

0.383

(n =1)

+

2+

Âľm, 0% Fe
4.4 Âą 0.1

Exp. 8
(n =1)

IOCS, 250~500
Âľm, 0.13% Fe

2+

Ca

a

n = number of replicates, EPM = electrophoretic mobility, MER = effluent mass recovery, kd = deposition rate coefficient, and
means in a column with different lower case letters are significantly different (P < 0.05) using the Least Significant Difference
(LSD) method.

20

2.2.4. XDLVO Calculations
Surface energies of zoospores interacting with uncoated sand or IOCS surfaces
were calculated according to the extended Derjaguin-Landau-Verwey-Overbeek
(XDLVO) theory, including Lifshitz-van der Waals, electrical double layer, and Born
repulsion interactions. Detailed calculations are provided in the Supporting Information
S2. The XDLVO interaction energies determine zoospore attachment strength on grain
surfaces at the primary and second energy minima, quantified by attachment efficiencies
(Îą). Then, theoretical kd values and effluent recoveries were estimated to compare with
the experimental results, thus allowing the assessment of the contribution of zoospore
attachment on the grain surface (i.e., the solid-water interface) to overall zoospore
retention.

2.3. RESULTS AND DISCUSSION
2.3.1. Characterization of Porous Media and Zoospores
The prepared IOCS had a brownish color (Figure 2.S2) indicative of iron oxide
coating. A closer examination of the IOCS surface by SEM revealed increased surface
roughness compared with the uncoated sand due to nano-sized features of iron oxide
(Figure 2.S4), as confirmed by energy-dispersive X-ray spectroscopy (Figure 2.S5). The
determined elemental Fe content was 0.003% for the uncoated sand, 0.11–0.13% for the
IOCS of lower Fe content, and 0.23% for the IOCS of higher Fe content, respectively. At
a near neutral pH of 8.0–8.5, the surface of IOCS became less negatively charged with
increasing Fe content, and in CaCl2 solution, surface charge was even reversed to the
positive sign (Table 2.S1). The observed charge reversal likely resulted from either Ca2+

21

adsorption on or accumulation near the IOCS surface.61-63 At the lower pH of 3.8, surface
charges of zoospore and porous media were much less negative, and the IOCS surface
became positively charged, due to protonation of IOCS surface. As the isoelectric point
of quartz and iron oxide was typically 1.5–3.0 and 7.5–9.0, respectively,64 iron oxide
surface was less negatively charged than quartz sand at identical solution pH and IS
(Table 2.S1). On average the IOCS surface had much less electrostatic repulsion and
probably even attraction to the negatively charged zoospores (Table 2.1). Consequently,
iron oxide coating often serves as favorable retention sites for negatively charged
particles such as Escherichia coli and Cryptosporidium parvum oocysts.65-67
Representative SEM images of biflagellate and encysted P. capsici zoospores in
CaCl2 solutions at pH 7.2 and IS 10 mM are shown in Figure 2.1. More SEM images of
the zoospores in DI water and NaCl solutions are provided in Figure 2.S6 and 2.S7.
Biflagellate zoospores were of ovoid shape (Figure 2.1, 2.S6, and 2.S7) with major axis
of 8.2 Âą 1.0 Âľm, minor axis of 5.7 Âą 0.5 Âľm, aspect ratio of 1.4 Âą 0.2, and roundness of
0.7 Âą 0.1, as measured by ImageJ. During encystment, the zoospores lost two flagella,
and were transformed into nearly a spherical shape with diameter of 6.9 Âą 0.4 Âľm, aspect
ratio of 1.0 Âą 0, and roundness of 1.0 Âą 0, similar to other Phytophthora zoospores.46, 47
Figure 2.S6C and Figure 2.S7B showed the shape of the zoospore in transition after
losing two flagella and prior to formation of the spherical encysts. Encysted zoospores in
the testing suspensions primarily consisted of the spherical shape; the presence of the
ovoid-shaped transitory encysts were minor, and the biflagellate zoospores were almost
nonexistent (Figure 2.1 and 2.S6). The surface of encysted and biflagellate zoospores
appeared rough and covered with extracellular surface coats probably containing acidic

22

surface groups68, 69 and polysaccharides.70 Another important change during encystment
was reported to be the secretion of adhesive materials such as glycoprotein from
peripheral vesicles and the formation of the outer surface coating of the cysts.71-73
As expected, under identical solution pH and IS, both biflagellate and encysted P.
capsici zoospores were less negatively charged in CaCl2 solution than in NaCl solution
(Table 2.1), which likely resulted from Ca2+ adsorption on or accumulation near the
zoospore surface. Surface charges of colloids and collector grains determine the electrical
double layer force important to colloid retention in porous media.23

23

Figure 2.1. Representative SEM images of encysted (A and C) and biflagellate (B) P.
capsici zoospores in CaCl2 solutions at pH 7.2 and IS 10 mM.

24

2.3.2. Retention of Encysted Zoospores
Biflagellate zoospores encyst within a few hours even in a quiescent suspension
and it was expected that the zoospores in water flows over longer spatial and time scales
would be the cysts most of time. Thus, the majority of the experiments focused on the
encysted zoospores. The influences of physicochemical properties of porous media and
solution chemistry on the transport of zoospores are discussed along with the retention
mechanisms for P. capsici zoospores.
2.3.2.1. Effect of Iron Oxide Coating and Grain Size
The presence of iron oxide surface coating significantly enhanced the retention of
encysted zoospores under identical solution chemistry as shown Figure 2.2. Examination
of MER and kd values for Expt. 2 and 6 revealed that in the 500-804 Âľm fraction the
zoospore transport through uncoated sand was significantly greater than through IOCS in
Ca2+ background solution, but no significant differences were observed in Na+
background solution (Table 2.1). In the 250–500 µm faction for Expt. 3, 4, and 5, the
zoospores were significantly less retained in the uncoated sand than in IOCS in both Na+
and Ca2+ background solutions (Table 2.1). It was noted that Figure 2.2A showed a
greater zoospore retention in the IOCS of 0.22% Fe than the IOCS of 0.13% Fe supported
by post hoc multiple comparisons of kd values (Table 2.1), but the MER statistics did not
show the same. Because the MER data were not normally distributed, the post hoc tests for
MER by the Least Significant Difference (LSD) method might not be valid in some cases
and thus not discussed hereafter. My results showed that iron oxide surface generally
facilitates the retention of biocolloids, agreeing with previous studies for E. coli67 and
Cryptosporidium parvum oocysts.65,

66

This observation was also in line with the Îś-

25

potential measurements as discussed previously (Table 2.S1) and the XDLVO
calculations showing less repulsive energy profiles for the zoospore interacting with
IOCS (Table 2.S2 and Figure 2.S9). Hence, for soils with greater iron oxide contents such
as Spodosol, Oxisol and Ultisol, the transport of zoospores would be much more limited.
Similarly, engineered filter media coated with nano-sized iron oxide could also be
employed to increase the retention of zoospores.
Interestingly, there was no significant difference of zoospore retention between
the 250–500 µm and 500–804 µm fractions (i.e., Expt. 2 vs Expt. 4 and Expt. 3 vs Expt.
6, Table 2.1). The ratio of zoospore to collector diameter was 0.009–0.0276, which is
much greater than the threshold ratio of 0.003 under which significant straining would
occur.74-77 Therefore, straining played an important role in zoospore retention. However,
XDLVO calculations also suggested strong surface attachment of zoospores to uncoated
sand and IOCS with attachment efficiencies (α) ranging 0.996–1.000. Thus, the coupling
between chemical and physical factors is expected to influence distribution of retained
zoospores on grain surface and pore straining sites.78-81 Given the strong zoospore
retention from surface attachment, decreasing grain size did not show a significant effect
on zoospore retention.
2.3.2.2. Effect of Solution Chemistry
There was no significant difference in the transport of encysted zoospores through
uncoated sand and IOCS of 250–500 µm in either NaCl or CaCl2 background solution
(Table 2.1 and Figure 2.2). These observations were contrary to the decreased Îśpotentials of zoospores (Table 2.1) and grain surface (Table 2.S1). XDLVO calculations
suggested almost complete retention of encysted zoospores, due to either the deep

26

secondary minima or the absence of primary maxima (Figure 2.S9 and Table 2.S2).
Indeed, the estimated effluent recovery was 0–0.006 and much lower than most
experimental MER values in Table 2.1. Therefore, the XDLVO calculations could not
fully explain the zoospore transport. The deficiencies of XDLVO theory could be
explained by its limitations for biological colloids due to more complex surface
properties, and by its inability to account for colloid retention by pore straining.
Intriguingly, the kd value of the zoospores in the IOCS of 500-804 Âľm was significantly
higher in Ca2+ background solution than Na+ background solution. In this case, stronger
XDLVO attractive force and less colloid straining due to larger collector grain size are
expected. Because colloid retention is highly dependent on the coupled effect of surface
attachment and pore straining,78,

79, 82

it is likely that the increased contribution from

surface attachment in the 500-804 Âľm IOCS made it possible to observe the greater
retention in Ca2+ background solution. Indeed, Bradford et al.75 reported that the
contribution of straining to the retention of similarly-sized Cryptosporidium oocysts (5
Âľm) was decreased from 79% for 360-Âľm sand to 68% for 710-Âľm sand. Lowering
solution pH increased the retention of encysted zoospores (Figure 2.3) due to protonation
of both sand and zoospore surfaces and subsequently increased electrostatic attraction, as
observed for other microorganisms.65, 83 XDLVO calculations showed a mostly favorable
condition for zoospore deposition (Table 2.S2, Figure 2.S9), indicating that XDLVO
calculation agreed with the experimental results reasonably well, despite its inherent
limitations.
However, it should be noted that despite almost complete retention predicted by
XDLVO calculations and strong straining, a small percentage of encysted zoospores (<

27

5.9%, Table 2.1) still passed through the column. Therefore, the XDLVO theory and
straining could not fully explain the zoospore transport and retention behaviors. This was
likely because rough outer surfaces (including glycoproteins and polysaccharides) of the
zoospores provided steric repulsion that is not included in XDLVO calculations, and this
steric repulsion could reduce the zoospore retention.
Encysted zoospores were also found to be attached on the IOCS surface through
filamentous materials (Figure 2.4 and 2.S8). Encysting zoospores released adhesive
content, likely glycoproteins, from peripheral vesicles to coat the cell surface and anchor
the zoospores to a solid surface.71-73 While it was argued that this type of attachment
mechanism occurred in the early stage of encystment,72 this study showed that secreted
adhesive materials on the cell surface could still attach the zoospores to the solid surface
long after encystment, thus partly contributing to the zoospore retention in the porous
media. Overall, the retention of zoospores in the porous media was collectively controlled
by electrostatic, steric and adhesive surface interactions, and pore straining.

28

Figure 2.2. Breakthrough curves of encysted P. capsici zoospores in NaCl or CaCl2
solutions (pH 7.2 and IS 10 mM) through saturated columns packed with IOCS (250−500
µm) of varying iron oxide coatings (A), IOCS and uncoated sand of 250−500 µm (B) and
500−804 µm (C).

29

Figure 2.3. Breakthrough curves of encysted P. capsici zoospores through saturated columns packed with IOCS and uncoated
sand at solution pH 4.4.

30

Figure 2.4. SEM images of encysted P. capsici zoospore attached on the IOCS surface in the presence of Ca2+.

31

2.3.2.3. Comparison of Biflagellate and Encysted Zoospores
Because both biflagellate and encystedc zoospores of P. capsici could be present
in contaminated water, I examined their transport behaviors under otherwise identical
experimental conditions. In the coarse IOCS, the retention of biflagellate zoospores was
much less than that of encysted zoospores in NaCl solution (Figure 2.5), and the kd value
was 0.122 Âą 0.011 (Exp. 1, Table 2.1), relative to the kd value of 0.216 Âą 0.023 for the
encysted zoospores (Exp. 2 , Table 2.1). Thus, when the biflagellate zoospores were
initially released from the sporangia, they were suited for enhanced mobility in porous
media. This observation agrees with the study of Wilkinson et al.40, reporting that
Phytophthora megasperma motile zoospores were transported much further than its
nonmotile cysts in unsaturated sand, sandy clay loam, and loam soils. It could be argued
that this mobility difference likely resulted from avoidance of the retention sites (e.g., low
flow regions or pore straining sites) by motile zoospores.22, 40 It could also be due to the
lack of secreted adhesive materials such as glycoprotein on the outer surface of the
biflagellate zoospores,22 as the adhesion of zoospores on the IOCS surface by the
adhesives (Figure 2.4 and 2.S8) was identified as one of retention mechanisms. In the
presence of Ca2+, the mobility of biflagellate zoospores was significantly decreased and
indistinguishable from that of encysted zoospores (Table 2.1 and Figure 2.1). This could
result from the inhibitory effect of Ca2+ on the zoospore motility due to increased
encystment in the presence of Ca2+,84 which would result in lower motility and increased
adhesive surface coats. It is possible that these mechanisms collectively contributed to the
reduced retention of biflagellate zoospores.

32

Figure 2.5. Breakthrough curves of biflagellate and encysted P. capsici zoospores through saturated columns packed with
IOCS (500–804 µm).

33

2.4. ENVIRONMENTAL IMPLICATIONS
My findings suggest that the transport of the plant pathogen P. capsici zoospores
in porous media was collectively controlled by surface properties of zoospores and
porous media, solution chemistry, and pore straining. Because zoospore retention in ironoxide coated sand or at lower solution pH was significantly enhanced, limited zoospore
dispersal through iron-oxide rich (e.g., Spodosol, Ultisol, and Oxisol) or acidic soils is
expected. For encysted zoospores, their mobility in soils is low because of their low
transport in the coarse sand tested in this study. However, considering the high transport
potential of motile biflagellate zoospores even in iron-oxide coated sand, the motile phase
of the zoospores appeared to be a period of substantial dispersal and infection risk, likely
during the first several hours of rainfall or irrigation events. Therefore, strategies to
control the water-induced Phytophthora infection from the zoospores could focus on the
short period following the water events. Given that the mobility difference of motile and
nonmotile zoospores was not observed in Ca2+ background solution, one effective
strategy to reduce pathogen transport is to induce zoospore encystment by increasing Ca
concentrations in soil water via liming. Additionally, this study also indicate that it is
possible to design filter media effective in removing plant pathogens from irrigation
water, which could be highly beneficial to the recirculating greenhouse production
systems. For the easy and precise detection by optical density, I used high zoospore input
concentrations of 4.4 ± 2.2 × 105 zoospores mL−1, and filtration reduced the peak
zoospore effluent concentration to about 2,000–17,000 zoospores mL−1 (i.e., about 1.2–
2.1 Log reduction). Because water containing ≥ 5000 zoospores/mL could infect 100% of
pickling cucumbers (Cucumis sativus) at temperatures above 12 °C,85 the small

34

percentage of transported zoospores might still pose a substantial risk to susceptible
crops. Hence, future research should be directed to investigate the transport behaviors of
zoospores at low input concentrations. Also, this study investigated one type of plant
pathogen in model porous media, and future studies should be conducted to examine
other types of plant pathogens in more realistic conditions.

2.5. ACKNOWLEDGMENTS
This research was supported by American Floral Endowment, The Fred C.
Gloeckner Foundation, and Michigan State University AgBioResearch. The views and
opinions of the authors expressed herein do not necessarily reflect those of funding
organizations and Michigan State University. Mention of tradenames or commercial
products does not constitute endorsement or recommendation for use.

35

APPENDIX

36

APPENDIX
SUPPORTING INFORMATION
S1. Preparation and Characterization of P. capsici Zoospores and Porous Media
S1.1. Preparation of Zoospore Suspension
To produce P. capsici zoospores, the culture was grown on V-8 agar (UCV8),
which consisted of 840 mL distilled water, 160 mL unclarified V8 juice, 30 mM CaCO3,
and 1.5% agar. The isolate was maintained on UCV8 at 25°C under continuous
fluorescent lighting. After 7–8 days, sterile distilled water was added to the culture, P.
capsici sporangia were scraped from the surface of the agar, and the resulting suspension
was placed into a 50- mL centrifuge tube. The tube was incubated at 4°C for 30–45
minutes and then at room temperature for 30 minutes to allow for zoospore release from
sporangia. Since zoospores are negatively geotropic, a stock zoospore suspension was
obtained by taking the top portion of the suspension, which excluded solid growth media,
hyphal fragments, and sporangia. To determine the concentration of the zoospore
suspension, a 1-mL aliquot was placed into a 1.7-mL microcentrifuge tube, vortexed for
70 seconds to induce zoospore encystment, and a 10-ÂľL aliquot was pipetted onto a clean
hemacytometer for counting (Bright-Line, Hausser Scientific, Horsham PA).
S1.2. SEM Imaging of Zoospores
To observe the zoospores by scanning electron microscope (SEM), 8 drops of the
zoospore suspension was mixed with an equal quantity of 4% glutaraldehyde buffered at
pH 7.4 with 0.1 M sodium phosphate. Fixation was allowed to proceed for 1 hour at 4°C.
A 12-mm round glass coverslip coverslip was coated with Poly-L-Lysine (Sigma Aldrich
P1399) by pressing a drop of 1% Poly-L-Lysine solution downward on a plastic petri dish

37

for 10 minutes and then gentle rinsing with several drops of water. One drop of the
zoospores fixed in suspension was placed on the wet Poly-L-Lysine coated coverslip
surface and settled for 10 minutes. The coverslip was then gently rinsed with several
drops of water, sequentially immersed in 25%, 40%, 50%, 60%, 75%, and 95% ethanol
solution for 10 minutes, and finally placed in 100% ethanol for 10 minutes for three
times. The samples were dried in a critical point dryer (Balzers Model 010, Balzers
Union Ltd., Balzers, Liechtenstein) using liquid CO2 as the transitional fluid, mounted on
aluminum stubs with carbon suspension cement (SPI Supplies, West Chester, PA), coated
with osmium (15 nm thickness) in a NEOC-AT osmium coater (Meiwafosis Co., Ltd.,
Osaka, Japan), and finally examined by a SEM with cold field emission electron emitter
(JEOL JSM-7500F, JEOL Ltd, Tokyo, Japan).

Figure 2.S1. Observed P. capsici zoospore number concentrations in relation to
absorbance.
38

S1.3. Iron Oxide Coating of Sand
To produce iron oxide coated sand, 200 gram of clean sand was added to a
mixture of 200-mL 0.05 M ferrous chloride solution and 40-mL 1 M NaHCO3 solution.
The slurry was stirred for 1.5 hr with magnetic bar to introduce oxygen into the
suspension and achieve uniform coating. During this process, Fe (II) in the slurry was
gradually oxidized to Fe (III), as shown by the color changes in Figure 2.S2B. After 1.5
hours of mixing, the coated sand was washed using copious amount of DI water, and then
oven dried at 110 °C for 24 hours. Finally, a reddish-brown coating was formed (Figure
2.S2C). This procedure was repeated four times in total in order to increase Fe content of
the coated sands. To create IOCS with a greater iron content, the mixture was stirred at a
higher speed with a stirrer (RW11, IKA, Germany) for a longer time. To determine iron
content, IOCS was soaked in 2.0 M HCl for 48 hours at room temperature until coated
iron was completely dissolved. Iron concentration in the prepared solutions was measure
by atomic absorption spectroscopy (AAnalyst 400, PerkinElmer, Waltham, MA). To
measure the Îś-potentials of IOCS and uncoated sand surfaces, a 5-gram sand sample was
ultrasonicated in 15 mL deionized (DI) water for 30 minutes and then vortexed to resuspend sand particles prior to the 206-min settling. The top 5-cm suspension was taken
for the Îś-potential measurements. This suspension approximately contained sand colloids
smaller than 2 µm, assuming the Stokes’ law and particle density of 2.65 g/cm3. Each ζpotential measurement was repeated six times and the average value was calculated.

39

B.

0 hr

0.5 hr

1.5 hr

C.
1

Repeat 1time
1

Repeat 2 times

Repeat 3 times

Repeat 4 times

Figure 2.S2. Schematic of preparation procedure for iron oxide coated sand (IOCS) (A),
and temporal visual changes of the preparation slurry (B) and IOCS (C).

40

Figure 2.S3. Observed and fitted breakthrough curves of bromide tracer through saturated
column packed with uncoated sand (250−500 µm).

41

Figure 2.S4. SEM images of Ottawa sand (A: 10,000 and C: 50,000) and iron oxide coated sand (B: 10,000 and D:
50,000) of 250–500 µm.

42

A

B

Figure 2.S5. Energy-dispersive X-ray spectra of uncoated (A) and iron oxide coated sand
(B).

43

Table 2.S1. Electrophoretic Mobility (EPM) and Îś-potential of Uncoated and Iron Oxide Coated Sand.
Uncoated sand
0.13% Fe IOCS
0.22% Fe IOCS
EPM
EPM
EPM
Solution chemistry
pH
Îś-potential
Îś-potential
Îś-potential
(µm cm s−1
(µm cm s−1
(µm cm s−1
(mV)
(mV)
(mV)
V−1)
V−1)
V−1)
0.4 mM NaHCO3
−43.8
−1.41
−18.0
−0.62
−7.9
8.5 −3.43 ± 0.16
+ 9.6 mM NaCl
Âą 2.1
Âą0.09
Âą1.2
Âą0.04
Âą0.6
0.4 mM NaHCO3
−12.1
0.28
3.6
0.58
7.3
8.0 −0.95 ± 0.04
+ 3.2 mM CaCl2
Âą 0.5
Âą0.03
Âą0.4
Âą8.35
Âą0.1
0.4 mM CH3COONa +
−23.8
1.35
17.2
2.96
37.8
2.6 mM CH3COOH + 9.6 3.8 −1.86 ± 0.09
Âą 1.2
Âą0.04
Âą0.5
Âą0.08
Âą1.0
mM NaCl
0.4 mM CH3COONa +
−8.9
1.79
22.8
3.40
43.4
2.6 mM CH3COOH + 3.2 3.8 −0.70 ± 0.04
Âą 0.5
Âą0.04
Âą0.6
Âą0.15
Âą1.9
mM CaCl2

44

Figure 2.S6. SEM images of encysted (A, C and D) and biflagellate P. capsici zoospores
(B) in deionized water.

45

Figure 2.S7. SEM images of biflagellate (A) and encysted (B) P. capsici zoospores in
NaCl solution at pH 7.2 and ionic strength 10 mM.

46

Figure 2.S8. SEM images of encysted P. capsici zoospores attached on iron oxide coated surface in the presence of Na+.

47

S2. XDLVO and Îątheory and kd theory Calculations
The

extended

Derjaguin-Landau-Verwey-Overbeek

(XDLVO)

interaction

energies, including Lifshitz-van der Waals (  LW ) , electrical double layer (  EL ), and
Born repulsion (  BR ) interactions, were calculated for a zoospore interacting with the
solid-water interface (SWI), i.e., uncoated sand or IOCS surface. The total XDLVO
interaction energy (ÎŚXDLVO) was determined as a function of separation distance (x):

 XDLVO ( x)   LW ( x)   EL ( x)   BR ( x)

(2.S1)

The  LW ,  EL , and  BR were calculated via:
 LW ( x)  

A132 ap
6x

1  14 x / c 1

(2.S2)86



1  exp(x) 
2
2


 EL ( x)   0 a p 2 1 2 ln 

(



)
ln
1

exp(

2

x
)

1
2

1  exp(x) 



(S3)87,

88

 BR ( x) 

A132 6
7560

 8a p  x
6a p  x 



7
x 7 
 (2a p  x)

(2.S3)89

where A132 is the Hamaker constant of the zoospore interacting with the SWI, ap is the
zoospore radius (i.e., 3.5 Âľm), Îťc is the van der Waals interaction characteristic
wavelength (i.e., about 100 nm), Îľ is the dielectric constant of the medium (i.e., 80.1 for
water at 293.15 K), ε0 is the vacuum permittivity (8.854 × 10−12 C2 N−1 m−2), ψ1 and ψ2
are the respective surface potential of the zoospore and the SWI, Îş is the reciprocal
electric double layer thickness (the Debye length [κ−1]), and σ (0.5 nm) is the collision
diameter. The A132 value for the zoospore is unknown, therefore, the A132 of 6.5 × 10−21 J
was taken from the literature values widely used for bacteria and Cryptosporidium
oocysts.65, 90, 91 I used the Îś-potentials in place of the surface potential.92 The XDLVO
48

calculations were performed for the encysted zoospores. The Îś-potentials of the encysted
zoospores in Table 2.1 under identical solution chemistry were averaged over several
experimental sets, as shown in Table 2.S2. The Îś-potentials of the uncoated sand and
IOCS of 0.13% Fe content as listed in Table 2.S2 were used to illustrate the XDLVO
energy of the zoospore interacting with the sand and IOCS surfaces. The calculated
XDLVO energies were normalized with kT where k is Boltzman constant (1.381 × 10−23 J
K−1) and T is temperature in Kelvin. The XDLVO primary energy maximum (Φmax) and
second energy minimum (ÎŚ2min) are listed in Table 2.S2 and the energy profiles are
shown in Figure 2.S9. Theoretical attachment efficiency (Îą) is calculated from a Maxwell
model that includes colloid deposition in secondary energy minimum.93, 94

 1 


 2 min

4



1/ 2

E 2 exp(  E 2 )dE
(2.S4)

where E2 is the particle kinetic energy normalized by kT, and ΔΦ is the sum of Φmax and
ÎŚ2min. The calculated Îą values are provided in Table 2.S2 below.
Table 2.S2. XDLVO Calculations and Attachment Efficiency for an Encysted Zoospore
Interacting with the Solid-Water Interface (SWI).a
Cation Îś-potential (mV)
ÎŚmax ÎŚ2min
Îą
Zoospore-SWI
pH
type
(kT)
(kT)
Zoospore SWI
Zoospore-Sand
High pH Na+
−18.0
−43.8
1770 6.6
0.996
Zoospore-Sand
(7.2–8.5) Ca+
−12.1
−12.1
137
12.0
1.000
Zoospore-IOCS
Na+
−18.0
−18.0
675
8.7
0.999
+
b
Zoospore-IOCS
Ca
−12.1
3.6
fav
fav
1.000
Zoospore-Sand
Low pH Na+
−9.9
−23.8
301
9.8
1.000
+
Zoospore-Sand
(3.8–4.4) Ca
−7.8
−8.9
fav
fav
1.000
Zoospore-IOCS
Na+
−9.9
−17.2
fav
fav
1.000
+
Zoospore-IOCS
Ca
−7.8
−22.8
fav
fav
1.000
a
ÎŚmax = primary energy maximum, ÎŚ2min = second energy minimum, Îątheory = theoretical
attachment efficiency; b fav indicate the favorable condition with the absence of ÎŚmax.
As shown in Table 2.S2 and Figure 2.S9, at pH 7.2-8.5 the zoospore-sand
interaction was characterized by high ÎŚmax and appreciable ÎŚ2min, whereas the zoospore49

IOCS interaction had lower ÎŚmax and deeper ÎŚ2min in the presence of Na+, and no ÎŚmax in
the presence of Ca2+. At pH 3.8–4.4, only the zoospore-sand interaction had Φmax and
ÎŚ2min in the presence of Na+, whereas all other interactions had no ÎŚmax. Because of either
the sizable ÎŚ2min or the absence of ÎŚmax, the calculated Îą was essentially 1, suggesting
complete retention of the zoospores by the porous media.
Additionally, theoretical deposition rate coefficient (kd theory) can be estimated as:95

kd theory 

3 (1  f )
v 0
2 dc

(2.S5)

where dc is the effective collector diameter, f is the porosity (0.35), v is the pore water
velocity (0.77 cm min−1), and η0 is the single-collector contact efficiency calculated by
the Tufenkji and Elimelech equation.95 The effective collector diameter was 375 Âľm for
the 250–500 µm fraction and 652 µm for the 500–804 µm fraction, respectively. The
zoospore density was 1.075 g cm−3 similar to Cryptosporidium parvum oocysts.65 The
calculated η0 was 0.0275–0.0333, and the kd

theory

was estimated to be 0.316–0.667.

Combining with Eq. (2), the theoretical effluent mass recovery (MER) was calculated to
be 0–0.006, again suggesting almost complete retention

50

S2. Column Breakthrough Experiments Raw Data a
Table 2.S3. Expt. 1: Biflagellate zoospores, IOCS, 500–804 µm, 0.11% Fe, pH 7.2 ± 0.2
Replicate 1
Replicate 2
Na+
Ca2+
Na+
Ca2+
𝜃 = 0.35
𝜃 = 0.35
𝜃 = 0.35
𝜃 = 0.35
𝑣 = 0.28 cm m-1
𝑣 = 0.27 cm m-1
𝑣 = 0.27 cm m-1
𝑣 = 0.8 cm m-1
Input PV = 1.8
Input PV = 1.9
Input PV = 1.9
Input PV = 1.8

a

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.001

0.000

0.002

0.000

0.000

0.295

0.000

0.316

0.000

0.313

0.000

0.303

0.000

0.595

0.006

0.634

0.000

0.628

0.000

0.610

0.000

0.894

0.013

0.953

0.000

0.942

0.001

0.916

0.001

1.194

0.026

1.272

0.004

1.257

0.015

1.222

0.005

1.493

0.077

1.591

0.006

1.572

0.119

1.529

0.008

1.793

0.159

1.910

0.009

1.887

0.223

1.835

0.007

2.093

0.307

2.229

0.021

2.202

0.212

2.142

0.011

2.392

0.220

2.548

0.057

2.517

0.149

2.448

0.013

2.692

0.053

2.866

0.030

2.831

0.038

2.754

0.008

2.992

0.010

3.185

0.007

3.146

0.004

3.061

0.000

3.291

0.006

3.504

0.003

3.461

0.002

3.367

0.000

3.591

0.004

3.823

0.004

3.776

0.001

3.674

0.000

3.890

0.006

4.142

0.002

4.091

0.001

3.980

0.000

4.190

0.004

4.461

0.003

4.405

0.000

4.286

0.000

4.490

0.003

4.780

0.003

4.720

0.001

4.593

0.000

4.789

0.000

5.098

0.003

5.035

0.001

4.899

0.000

5.089

0.000

5.417

0.002

5.350

0.001

5.206

0.000

5.389

0.000

5.736

0.001

5.665

0.001

5.512

0.000

5.688

0.001

6.055

0.003

5.980

0.001

5.818

0.000

5.988

0.000

6.374

0.002

6.294

0.001

6.125

0.000

6.287

0.000

6.693

0.001

6.609

0.000

6.431

0.000

6.587

0.000

7.012

0.003

6.924

0.000

6.738

0.000

6.887

0.000

7.330

0.002

7.239

0.000

7.044

0.000

PV = Pore volumes, θ = volumetric water content, and v = average pore water velocity.

51

Table 2.S4. Expt. 2: Encysted zoospores, IOCS, 500–804 µm, 0.11% Fe, pH 7.2 ± 0.2
Replicate 1
Replicate 2
Na+

Ca2+

Na+

Ca2+

𝜃 = 0.35

𝜃 = 0.35

𝜃 = 0.35

𝜃 = 0.35

𝑣 = 0.28 cm m-1

𝑣 = 0.28 cm m-1

𝑣 = 0.28 cm m-1

𝑣 = 0.27 cm m-1

Input PV = 1.8

Input PV = 1.8

Input PV = 1.8

Input PV = 1.9

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.298

0.002

0.295

0.000

0.296

0.001

0.302

0.000

0.601

0.000

0.595

0.000

0.597

0.003

0.610

0.002

0.903

0.014

0.895

0.007

0.898

0.009

0.918

0.006

1.206

0.027

1.195

0.016

1.198

0.011

1.226

0.005

1.509

0.029

1.495

0.016

1.499

0.016

1.534

0.008

1.811

0.032

1.795

0.015

1.800

0.014

1.842

0.009

2.114

0.033

2.095

0.014

2.101

0.023

2.150

0.010

2.417

0.027

2.395

0.015

2.402

0.025

2.458

0.011

2.719

0.010

2.695

0.006

2.702

0.019

2.766

0.004

3.022

0.002

2.995

0.006

3.003

0.012

3.074

0.001

3.325

0.003

3.295

0.000

3.304

0.009

3.382

0.002

3.627

0.006

3.595

0.000

3.605

0.008

3.690

0.001

3.930

0.002

3.895

0.000

3.905

0.008

3.998

0.000

4.233

0.000

4.195

0.000

4.206

0.016

4.306

0.001

4.535

0.000

4.495

0.000

4.507

0.009

4.614

0.000

4.838

0.002

4.795

0.000

4.808

0.009

4.922

0.000

5.141

0.002

5.094

0.000

5.108

0.009

5.230

0.001

5.444

0.000

5.394

0.000

5.409

0.002

5.538

0.002

5.746

0.000

5.694

0.000

5.710

0.004

5.846

0.000

6.049

0.000

5.994

0.000

6.011

0.005

6.154

0.000

6.352

0.000

6.294

0.000

6.312

0.006

6.462

0.000

6.654

0.000

6.594

0.000

6.612

0.005

6.770

0.002

6.957

0.000

6.894

0.000

6.913

0.007

7.078

0.003

continue

52

Table 2.S5. Expt. 2: Encysted zoospores, IOCS, 500–804 µm, 0.11% Fe, pH 7.2 ± 0.2
Replicate 3
Na+

Ca2+

𝜃 = 0.35

𝜃 = 0.35
-1

𝑣 = 0.27 cm m

𝑣 = 0.26 cm m-1

Input PV = 1.9

Input PV = 1.9

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.001

0.000

0.000

0.304

0.000

0.316

0.000

0.613

0.004

0.636

0.001

0.922

0.001

0.956

0.002

1.231

0.007

1.276

0.006

1.540

0.007

1.596

0.006

1.849

0.015

1.916

0.010

2.158

0.021

2.237

0.013

2.467

0.025

2.557

0.018

2.776

0.017

2.877

0.014

3.085

0.007

3.197

0.004

3.394

0.003

3.517

0.000

3.703

0.000

3.837

0.001

4.012

0.001

4.157

0.002

4.321

0.002

4.478

0.001

4.630

0.005

4.798

0.003

4.939

0.005

5.118

0.000

5.248

0.000

5.438

0.000

5.557

0.000

5.758

0.000

5.867

0.000

6.078

0.000

6.176

0.000

6.399

0.000

6.485

0.000

6.719

0.001

6.794

0.000

7.039

0.000

7.103

0.000

7.359

0.000

continue

53

Table 2.S6. Expt. 3: Encysted zoospores, Sand, 250~500 Âľm, 0% Fe, pH 7.2 Âą 0.2
Replicate 1
Replicate 2
Na+

Ca2+

Na+

Ca2+

𝜃 = 0.35

𝜃 = 0.36

𝜃 = 0.35

𝜃 = 0.36

𝑣 = 0.28 cm m-1

𝑣 = 0.28 cm m-1

𝑣 = 0.27 cm m-1

𝑣 = 0.29 cm m-1

Input PV = 1.8

Input PV = 1.8

Input PV = 1.9

Input PV = 1.8

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.001

0.000

0.000

0.000

0.002

0.000

0.000

0.296

0.000

0.288

0.000

0.315

0.000

0.286

0.000

0.596

0.000

0.581

0.001

0.633

0.000

0.578

0.000

0.896

0.007

0.875

0.013

0.952

0.015

0.869

0.008

1.197

0.045

1.168

0.054

1.270

0.057

1.160

0.049

1.497

0.051

1.462

0.061

1.588

0.061

1.451

0.060

1.798

0.054

1.755

0.063

1.907

0.064

1.743

0.058

2.098

0.054

2.049

0.065

2.225

0.065

2.034

0.061

2.398

0.057

2.342

0.067

2.544

0.068

2.325

0.063

2.699

0.042

2.635

0.050

2.862

0.046

2.616

0.045

2.999

0.004

2.929

0.011

3.181

0.014

2.908

0.007

3.300

0.006

3.222

0.010

3.499

0.003

3.199

0.000

3.600

0.003

3.516

0.009

3.817

0.004

3.490

0.001

3.900

0.005

3.809

0.000

4.136

0.004

3.782

0.001

4.201

0.003

4.103

0.002

4.454

0.000

4.073

0.001

4.501

0.004

4.396

0.003

4.773

0.002

4.364

0.000

4.802

0.002

4.689

0.005

5.091

0.002

4.655

0.000

5.102

0.000

4.983

0.002

5.410

0.002

4.947

0.000

5.402

0.000

5.276

0.003

5.728

0.003

5.238

0.001

5.703

0.000

5.570

0.002

6.046

0.004

5.529

0.001

6.003

0.000

5.863

0.003

6.365

0.003

5.820

0.001

6.304

0.000

6.157

0.003

6.683

0.000

6.112

0.002

6.604

0.000

6.450

0.004

7.002

0.002

6.403

0.001

6.904

0.000

6.743

0.003

7.320

0.000

6.694

0.001

continue

54

Table 2.S7. Expt. 3: Encysted zoospores, Sand, 250~500 Âľm, 0% Fe, pH 7.2 Âą 0.2
Replicate 3
Na+

Ca2+

𝜃 = 0.35

𝜃 = 0.36
-1

𝑣 = 0.28 cm m

𝑣 = 0.29 cm m-1

Input PV = 1.8

Input PV = 1.8

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.297

0.000

0.290

0.000

0.598

0.001

0.583

0.002

0.898

0.007

0.876

0.003

1.199

0.039

1.170

0.033

1.500

0.041

1.463

0.049

1.800

0.042

1.757

0.049

2.101

0.042

2.050

0.044

2.402

0.048

2.343

0.043

2.703

0.035

2.637

0.019

3.003

0.011

2.930

0.004

3.304

0.010

3.224

0.000

3.605

0.005

3.517

0.000

3.906

0.007

3.810

0.000

4.206

0.005

4.104

0.000

4.507

0.004

4.397

0.000

4.808

0.004

4.691

0.000

5.108

0.004

4.984

0.000

5.409

0.000

5.277

0.000

5.710

0.000

5.571

0.000

6.011

0.000

5.864

0.000

6.311

0.000

6.158

0.000

6.612

0.000

6.451

0.000

6.913

0.000

6.744

0.000

continue

55

Table 2.S 8. Expt. 4: Encysted zoospores, IOCS,250~500 Âľm, 0.13% Fe, pH 7.2 Âą 0.2
Replicate 1
Replicate 2
Na+

Ca2+

𝜃 = 0.36

Na+

𝜃 = 0.36
-1

Ca2+

𝜃 = 0.36
-1

𝜃 = 0.36
-1

𝑣 = 0.28 cm m

𝑣 = 0.29 cm m

𝑣 = 0.28 cm m

𝑣 = 0.28 cm m-1

Input PV = 1.8

Input PV = 1.8

Input PV = 1.8

Input PV = 2.0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.296

0.001

0.290

0.000

0.293

0.002

0.343

0.000

0.597

0.002

0.584

0.000

0.590

0.004

0.675

0.000

0.897

0.002

0.878

0.000

0.887

0.004

1.007

0.000

1.197

0.002

1.171

0.003

1.184

0.007

1.339

0.005

1.497

0.015

1.465

0.018

1.480

0.019

1.670

0.015

1.798

0.026

1.759

0.029

1.777

0.030

2.002

0.030

2.098

0.035

2.053

0.034

2.074

0.036

2.334

0.040

2.398

0.035

2.347

0.039

2.371

0.036

2.666

0.027

2.698

0.022

2.641

0.037

2.667

0.028

2.997

0.005

2.998

0.000

2.935

0.017

2.964

0.011

3.329

0.000

3.299

0.000

3.229

0.007

3.261

0.003

3.661

0.000

3.599

0.000

3.523

0.004

3.558

0.005

3.993

0.000

3.899

0.007

3.816

0.003

3.854

0.004

4.325

0.000

4.199

0.007

4.110

0.005

4.151

0.005

4.656

0.000

4.499

0.000

4.404

0.000

4.448

0.004

4.988

0.000

4.800

0.000

4.698

0.000

4.745

0.004

5.320

0.000

5.100

0.000

4.992

0.000

5.041

0.007

5.652

0.000

5.400

0.000

5.286

0.000

5.338

0.006

5.983

0.000

5.700

0.000

5.580

0.000

5.635

0.003

6.315

0.000

6.001

0.000

5.874

0.000

5.932

0.003

6.647

0.000

6.301

0.000

6.168

0.000

6.229

0.001

6.979

0.000

6.601

0.000

6.462

0.000

6.525

0.003

7.310

0.000

6.901

0.000

6.755

0.000

6.822

0.003

7.642

0.000

continue

56

Table 2.S 9. Expt. 4: Encysted zoospores, IOCS,250~500 Âľm, 0.13% Fe, pH 7.2 Âą 0.2
Replicate 3
Na+

Ca2+

𝜃 = 0.34

𝜃 = 0.34
-1

𝑣 = 0.27 cm m

𝑣 = 0.26 cm m-1

Input PV = 1.9

Input PV = 1.9

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.305

0.001

0.316

0.000

0.616

0.000

0.636

0.000

0.927

0.000

0.956

0.000

1.237

0.001

1.276

0.000

1.548

0.008

1.595

0.010

1.859

0.016

1.915

0.016

2.170

0.019

2.235

0.017

2.480

0.021

2.555

0.022

2.791

0.012

2.874

0.014

3.102

0.002

3.194

0.004

3.413

0.000

3.514

0.001

3.723

0.000

3.834

0.001

4.034

0.000

4.153

0.000

4.345

0.000

4.473

0.000

4.656

0.000

4.793

0.000

4.966

0.000

5.113

0.000

5.277

0.000

5.432

0.000

5.588

0.000

5.752

0.004

5.899

0.000

6.072

0.004

6.209

0.002

6.392

0.000

6.520

0.000

6.711

0.002

6.831

0.000

7.031

0.003

7.142

0.000

7.351

0.000

continue

57

Table 2.S 10. Expt. 5: Encysted zoospores, IOCS, 250~500 Âľm, 0.22% Fe, pH 7.2 Âą 0.2
Replicate 1
Replicate 2
Na+

Ca2+

𝜃 = 0.35

Na+

𝜃 = 0.35
-1

Ca2+

𝜃 = 0.35
-1

𝜃 = 0.35
-1

𝑣 = 0.23 cm m

𝑣 = 0.27 cm m

𝑣 = 0.26 cm m

𝑣 = 0.28 cm m-1

Input PV = 1.9

Input PV = 1.9

Input PV = 2.0

Input PV = 1.8

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.001

0.000

0.003

0.000

0.000

0.000

0.000

0.355

0.000

0.305

0.000

0.324

0.000

0.296

0.000

0.716

0.000

0.615

0.000

0.652

0.003

0.597

0.001

1.076

0.000

0.924

0.000

0.980

0.000

0.898

0.000

1.437

0.002

1.234

0.003

1.308

0.000

1.198

0.000

1.797

0.005

1.544

0.005

1.636

0.001

1.499

0.001

2.158

0.010

1.854

0.007

1.964

0.004

1.800

0.001

2.519

0.011

2.163

0.003

2.292

0.009

2.100

0.001

2.879

0.005

2.473

0.005

2.620

0.014

2.401

0.006

3.240

0.003

2.783

0.001

2.948

0.013

2.702

0.009

3.600

0.005

3.092

0.004

3.276

0.006

3.002

0.002

3.961

0.019

3.402

0.000

3.604

0.005

3.303

0.001

4.322

0.000

3.712

0.002

3.931

0.002

3.604

0.000

4.682

0.000

4.022

0.005

4.259

0.001

3.904

0.000

5.043

0.004

4.331

0.008

4.587

0.000

4.205

0.000

5.403

0.001

4.641

0.003

4.915

0.000

4.506

0.000

5.764

0.001

4.951

0.003

5.243

0.000

4.806

0.001

6.125

0.005

5.260

0.004

5.571

0.000

5.107

0.000

6.485

0.000

5.570

0.003

5.899

0.000

5.408

0.000

6.846

0.000

5.880

0.003

6.227

0.000

5.708

0.000

7.206

0.000

6.190

0.002

6.555

0.000

6.009

0.000

7.567

0.000

6.499

0.002

6.883

0.000

6.310

0.000

7.928

0.000

6.809

0.003

7.211

0.000

6.611

0.000

8.288

0.000

7.119

0.003

7.539

0.000

6.911

0.000

continue

58

Table 2.S 11. Expt. 6: Encysted zoospores, Sand, 500~804 Âľm, 0% Fe, pH 7.2 Âą 0.2
Replicate 1
Replicate 2
Na+

Ca2+

𝜃 = 0.35

Na+

𝜃 = 0.35
-1

Ca2+

𝜃 = 0.35
-1

𝜃 = 0.35
-1

𝑣 = 0.28 cm m

𝑣 = 0.25 cm m

𝑣 = 0.26 cm m

𝑣 = 0.27 cm m-1

Input PV = 1.8

Input PV = 2.0

Input PV = 2.0

Input PV = 1.9

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.001

0.000

0.002

0.000

0.000

0.000

0.001

0.301

0.000

0.324

0.000

0.317

0.001

0.304

0.000

0.606

0.000

0.653

0.000

0.640

0.001

0.614

0.001

0.911

0.017

0.982

0.009

0.964

0.016

0.924

0.009

1.216

0.046

1.311

0.038

1.288

0.043

1.234

0.031

1.521

0.056

1.640

0.049

1.612

0.052

1.544

0.041

1.826

0.052

1.969

0.047

1.935

0.049

1.854

0.049

2.131

0.053

2.298

0.053

2.259

0.049

2.164

0.048

2.436

0.051

2.628

0.051

2.583

0.047

2.474

0.048

2.741

0.035

2.957

0.037

2.907

0.031

2.784

0.034

3.046

0.012

3.286

0.006

3.231

0.001

3.094

0.013

3.351

0.001

3.615

0.000

3.554

0.000

3.404

0.008

3.656

0.000

3.944

0.000

3.878

0.000

3.714

0.008

3.961

0.000

4.273

0.000

4.202

0.000

4.024

0.006

4.266

0.000

4.602

0.000

4.526

0.000

4.334

0.008

4.571

0.000

4.931

0.000

4.849

0.000

4.644

0.002

4.876

0.000

5.261

0.000

5.173

0.000

4.954

0.003

5.181

0.000

5.590

0.000

5.497

0.000

5.264

0.002

5.486

0.000

5.919

0.001

5.821

0.000

5.575

0.002

5.791

0.000

6.248

0.001

6.144

0.000

5.885

0.001

6.096

0.000

6.577

0.000

6.468

0.000

6.195

0.002

6.401

0.000

6.906

0.001

6.792

0.000

6.505

0.001

6.706

0.000

7.235

0.000

7.116

0.000

6.815

0.002

7.011

0.000

7.564

0.000

7.440

0.000

7.125

0.000

continue

59

Table 2.S 12. Expt. 6: Encysted zoospores, Sand, 500~804 Âľm, 0% Fe, pH 7.2 Âą 0.2
Replicate 3
Na+

Ca2+

𝜃 = 0.35

𝜃 = 0.36
-1

𝑣 = 0.25 cm m

𝑣 = 0.28 cm m-1

Input PV = 2.0

Input PV = 1.9

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.326

0.002

0.319

0.000

0.658

0.000

0.636

0.007

0.989

0.007

0.952

0.003

1.320

0.020

1.269

0.016

1.651

0.027

1.586

0.026

1.982

0.023

1.903

0.024

2.313

0.023

2.219

0.028

2.645

0.022

2.536

0.022

2.976

0.013

2.853

0.019

3.307

0.000

3.170

0.002

3.638

0.000

3.486

0.000

3.969

0.000

3.803

0.000

4.301

0.000

4.120

0.000

4.632

0.000

4.437

0.000

4.963

0.000

4.753

0.000

5.294

0.000

5.070

0.000

5.625

0.000

5.387

0.000

5.956

0.000

5.704

0.000

6.288

0.000

6.020

0.000

6.619

0.000

6.337

0.000

6.950

0.000

6.654

0.000

7.281

0.000

6.971

0.000

7.612

0.000

7.287

0.000

continue

60

Table 2.S 13. Expt. 7: Encysted zoospores, Sand, 250~500 Âľm, 0% Fe, pH 4.4 Âą 0.1
Replicate 1
Na+

Ca2+

𝜃 = 0.34

𝜃 = 0.35
-1

𝑣 = 0.28 cm m

𝑣 = 0.27 cm m-1

Input PV = 1.8

Input PV = 1.9

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.301

0.001

0.308

0.002

0.606

0.008

0.620

0.001

0.911

0.006

0.932

0.000

1.217

0.003

1.244

0.003

1.522

0.012

1.556

0.004

1.827

0.009

1.869

0.006

2.133

0.004

2.181

0.003

2.438

0.004

2.493

0.007

2.743

0.007

2.805

0.012

3.049

0.008

3.118

0.000

3.354

0.000

3.430

0.000

3.659

0.003

3.742

0.000

3.964

0.001

4.054

0.000

4.270

0.000

4.366

0.000

4.575

0.005

4.679

0.000

4.880

0.009

4.991

0.000

5.186

0.003

5.303

0.000

5.491

0.006

5.615

0.000

5.796

0.006

5.927

0.000

6.101

0.006

6.240

0.000

6.407

0.005

6.552

0.000

6.712

0.004

6.864

0.000

7.017

0.007

7.176

0.000

continue

61

Table 2.S 14. Tracer(NaBr), sand 250~500 µm, IS = 0, 𝐶0 = 100 mg L-1
𝜃 = 0.35
𝑣 = 0.28 cm m-1
Input PV = 1.7
PV

𝐶/𝐶0

0.000

0.001

0.276

0.000

0.564

0.000

0.851

0.157

1.139

0.959

1.426

1.015

1.714

1.007

2.001

1.009

2.289

1.019

2.576

0.927

2.864

0.106

3.152

0.004

3.439

0.002

3.727

0.000

4.014

0.000

4.302

0.000

4.589

0.000

4.877

0.000

5.164

0.000

5.452

0.000

5.739

0.000

6.027

0.000

6.314

0.000

6.602

0.000

continue

62

Table 2.S 15. Expt. 8: Encysted zoospores, IOCS, 250~500 Âľm, 0.13% Fe, pH 4.4 Âą 0.1
Replicate 1
Na+

Ca2+

𝜃 = 0.34

𝜃 = 0.35
-1

𝑣 = 0.27 cm m

𝑣 = 0.27 cm m-1

Input PV = 1.8

Input PV = 1.8

PV

𝐶/𝐶0

PV

𝐶/𝐶0

0.000

0.000

0.000

0.000

0.298

0.008

0.301

0.000

0.601

0.006

0.606

0.000

0.905

0.000

0.912

0.005

1.208

0.000

1.217

0.002

1.511

0.005

1.523

0.003

1.815

0.003

1.828

0.000

2.118

0.003

2.134

0.000

2.422

0.000

2.440

0.000

2.725

0.001

2.745

0.000

3.029

0.000

3.051

0.000

3.332

0.000

3.356

0.000

3.635

0.000

3.662

0.000

3.939

0.000

3.968

0.000

4.242

0.000

4.273

0.000

4.546

0.000

4.579

0.000

4.849

0.000

4.884

0.000

5.152

0.000

5.190

0.001

5.456

0.000

5.495

0.000

5.759

0.000

5.801

0.000

6.063

0.000

6.107

0.000

6.366

0.000

6.412

0.000

6.669

0.000

6.718

0.000

6.973

0.000

7.023

0.000

continue

63

Figure 2.S9. Total XDLVO interaction energies of zoospore interacting with uncoated
sand surface or iron oxide coated sand (IOCS) surface: (a) primary energy minimum
(ÎŚ1min) and maximum (ÎŚmax) and (b) secondary energy minimum (ÎŚ2min).

64

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Resour. Res. 2006, 42, (12), W12S15.

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Torkzaban, S.; Bradford, S. A.; Walker, S. L., Resolving the coupled effects of
hydrodynamics and DLVO forces on colloid attachment in porous media. Langmuir
2007, 23, (19), 9652-9660.

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Bradford, S. A.; Torkzaban, S.; Walker, S. L., Coupling of physical and chemical
mechanisms of colloid straining in saturated porous media. Water Res. 2007, 41,
(13), 3012-3024.

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Zevi, Y.; Dathe, A.; Gao, B.; Zhang, W.; Richards, B. K.; Steenhuis, T. S.,
Transport and retention of colloidal particles in partially saturated porous media:
Effect of ionic strength. Water Resour. Res. 2009, 45, (12), W12403.

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Zevi, Y.; Gao, B.; Zhang, W.; Morales, V. L.; Cakmak, M. E.; Medrano, E. A.;
Sang, W.; Steenhuis, T. S., Colloid retention at the meniscus-wall contact line in an
open microchannel. Water Res. 2012, 46, (2), 295-306.

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Kim, H. N.; Walker, S. L.; Bradford, S. A., Coupled factors influencing the
transport and retention of Cryptosporidium parvum oocysts in saturated porous
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Kim, H. N.; Bradford, S. A.; Walker, S. L., Escherichia coli O157:H7 transport in
saturated porous media: Role of solution chemistry and surface macromolecules.
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and Infectivity of Phytophthora parasitica in Nutrient Irrigation Solutions.
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Algaecides on Phytophthora capsici Zoospore Infectivity. Plant Dis. 2010, 94, (1),
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particles and rough surfaces. J. Colloid Interface Sci. 2006, 298, (1), 50-58.

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chromatographic separation. AICHE J. 1976, 22, (2), 276-283.

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porous media:  Role of the secondary energy minimum. Environ. Sci. Technol.
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oocysts to quartz surfaces. Environ. Sci. Technol. 2004, 38, (24), 6839-6845.

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van Oss, C. J., Interfacial forces in aqueous media. 2nd ed.; CRC Taylor & Francis:
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efficiency in physicochemical filtration in saturated porous media. Environ. Sci.
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72

CHAPTER 3

FILTRATION OF PYTHIUM APHANIDERMATUM ZOOSPORES IN
RECYCLED IRRIGATION WATER TO CONTROL POINSETTIA DISEASE IN
GREENHOUSES

ABSTRACT
Pythium aphanidermatum incites crown and root rot and can be highly destructive
to floriculture greenhouse crops especially when recirculating irrigation water systems
are used. This study aimed to demonstrate a proof-of-concept using fast-flow filtration to
control Pythium root rot of poinsettia grown with ebb-and-flow and flood-floor irrigation
systems in commercial greenhouses. Two experiments were performed in a research
greenhouse to investigate the effect of filter media type (i.e., sand and activated carbon),
fungicide application (i.e., etridiozole) and pathogen inoculum source (i.e., infected
plants vs infested irrigation water). The fast-flow sand filtration with low water pressure
consistently removed Pythium aphanidermatum zoospores, and significant improvements
in root rot severity, height, biomass, and horticultural rating were observed for the plants
in the sand filter treatment, compared with the inoculated control plants. However, the
activated carbon filter removed essential nutrients from the irrigation water, resulting in
plant nutrient deficiency and consequently leaf chlorosis and reduced plant biomass,
height, and horticultural rating. The application of etridiozole did not completely prevent
root infection by P. aphanidermatum, but the plant biomass, height, and horticultural
rating were not negatively affected by the pathogen. P. aphanidermatum spread from
infected plants to healthy plants with the recycled irrigation water. Overall, the rapid sand
filter has the potential to limit P. aphanidermatum spread and the associated root rot in

73

greenhouse floriculture crops.
3.1. INTRODUCTION
Floriculture crops in the U.S. have an estimated wholesale value of $4.4 billion,
and include a diverse assortment of bedding plants, potted flowers, and nursery crops.
Poinsettias contributed a wholesale value of $140 million in 2015 and are one of the top
potted flowering plants in the U.S.1, 2 In the greenhouse production of floriculture crops,
recirculating irrigation systems have been widely adopted to lower water usage, and
conserve fertilizers that can otherwise be lost via discharge runoff.3-7 This is especially
true for greenhouses with large water use, i.e., 0.5–1 million gallons per day.8 Irrigation
systems with ebb-and-flow and flood-floor are also used to maximize production area and
decrease labor costs.9, 10 In this type of irrigation system, irrigation water is pumped from
a water reservoir to flood a floor at a specified water level for a desired duration, and then
drained back (often by gravity flow) to the reservoir for recycling in the next irrigation
event. Although recycling irrigation water offers many benefits to greenhouse growers,
plant pathogens can also be disseminated in the recycled irrigation water .10 Thus,
limiting pathogen transmission in the recirculating irrigation systems is critical to the
floriculture industry.
Pythium spp. and other water molds can be highly destructive to floriculture
crops, and spread readily in irrigation water.11-15 Pythium root rot causes plant wilting and
death and reduces horticultural quality.16 The pathogen can become established in a
greenhouse through infested soil and dust17, seedlings, cuttings, or other plant material
from propagation greenhouses,9 or infested surface water used for irrigation.3
Management of Pythium spp. is particularly challenging for potted plants; frequent

74

irrigation and high moisture levels are ideal for the reproduction and transmission of this
pathogen.18 The high porosity of peat potting media may also facilitate the movement of
zoospores, which are an important type of Pythium spp. inoculum.19 Thus, Pythium spp.
could be rapidly disseminated in a greenhouse via ebb and flow flood floor production
systems,20 leading to crop damage and loss, thus requiring proactive management
strategies.
Fungicide application is a common and important strategy to limit Pythium root
rot in greenhouse production.21 Currently, the primary fungicides used for Pythium root
rot control are etridiazole (Terrazole, OHP, Mainland, PA) and mefenoxam (Subdue
Maxx, Syngenta Crop Protection, Greensborough, NC).21,

22

Etridiazole effectively

reduced Pythium root rot in poinsettia and easter lily when applied as a soil drench.22-24
Also, etridiazole is one of the few commercial fungicides that is labeled for use in the
ebb-and-flow and flood-floor irrigation systems.25 Mefenoxam can also limit crop loss
from Pythium root rot.26 However, resistance to mefenoxam has developed in greenhouse
populations of Pythium spp., partly due to repeated fungicide use.21,

26, 27

Failure to

control Pythium diseases using mefenoxam has been reported in greenhouses,21, 28 and
resistant isolates were detected in surface water used for irrigation.29 Fungicide resistance
has become a limiting factor for the control of Pythium crown and root rot; alternative
strategies for pathogen control in irrigation water are needed.30
Management of pathogens in recycled irrigation water is challenging. Ultra-violet
radiation, heat treatment, chemical disinfection, ozonation, and filtration have been used
to remove pathogens from irrigation water with varying degrees of success.10, 15 Many of
these methods are cost-prohibitive to install and operate in commercial greenhouses. In

75

contrast, filtration is a low-cost method that could potentially disinfest irrigation water by
the physical removal of pathogens using granular porous media (e.g., sand) or membrane
filters.15 Membrane filtration can effectively remove zoospores if the membrane pore size
is small enough to retain the motile zoospores that have a pleomorphic cell membrane.31
Membrane filters with pore sizes of 1 and 5 Âľm were able to effectively remove the
Pythium zoospores from recirculating irrigation water in laboratory tests.32 However, it is
unknown whether this could be transferable to greenhouse settings. Diplanetism (i.e.,
where a zoospore encysts and releases a smaller motile zoospore) could decrease the
efficacy of membrane filters,33 although the occurrence of diplantetism in commercial
greenhouses is unknown. An additional challenge with membrane filters is frequent
leakage, and membrane clogging and fouling

10, 32

, resulting in increased maintenance

cost and decreased performance over time.
In contrast, deep-bed filtration (e.g., sand filtration) is a cost-effective alternative
in terms of construction, operation and maintenance. Slow filtration with granular
materials has been studied as a means to remove Pythium spp. from the greenhouse
irrigation water in the 1970s.34 However, it isn’t widely used in commercial U.S.
greenhouses due to the slow water flow rate (i.e., 100–300 L/m2/h)10 that prohibits the
movement of a large volume of water to multiple greenhouse ranges in an acceptable
time period.15 Previous studies focused on the ability of slow sand filtration in limiting
plant pathogens in irrigation water.10, 15, 35 The effectiveness of fast-flow filtration on
pathogen removal from irrigation water has not been well investigated. Additional data
could help to determine whether this technique could be adopted to manage irrigation
water in greenhouses.

76

The objective of this study was to investigate the ability of fast-flow filtration to
limit Pythium diseases for potted poinsettias in greenhouses with ebb-and-flow and floodfloor irrigation systems. Six small-scale ebb-and-flow recirculating irrigation systems
were constructed to simultaneously test the effect of filter media type (i.e., sand and
activated carbon), fungicide application (i.e., etridiazole), and pathogen transmission
mode (i.e., infected plants vs infested water). Poinsettia was used as a model crop
because of its popularity as a potted flower and the prevalence of Pythium outbreaks in its
production. This study was intended to show a proof-of-concept to use the fast-flow
filtration system in removing plant pathogens from recirculating irrigation water.

3.2. MATERIALS AND METHODS
3.2.1. Irrigation and Filtration Systems.
Six small-scale irrigation systems were constructed to simulate the ebb-and-flow
and flood-floor systems in greenhouse settings, consisting of an ebb-and-flow bench, an
optional pre-filter tank, an optional filter unit, and a holding tank (Figure 3.S1). Only one
holding tank was included in some of filter-free treatments, including the non-inoculated
and inoculated control treatment in the first experiment, and in the inoculated control and
“diseased plant” treatments in the second experiment. Otherwise, the pre-filter tank and
the holding tank were directly connected for the non-filtration treatments. The filter unit
was designed as shown in Figure 3.S2, and was packed with either sand (99.69% silica,
GranusilÂŽ ) or activated carbon (AC) (Filtrasorb 300, CalgonCarbon, USA). Particle size
distribution of the sand was 5.1% of 297-420 µm, 57.2% of 420-595 µm, 36.1% of 595841 µm, and 1.2 % of ≥ 841 µm. The effective size of AC particles was 0.8-1.0 mm.
Detailed description on the irrigation systems and filter units is provided in Supporting
77

Information S1. The irrigation systems allowed for automatic irrigation of potted plants
placed in the bench via flooding according to a pre-designated schedule. These irrigation
systems were subsequently used in two greenhouse experiments.
3.2.2. Pathogen Culture and Inoculum.
Pythium aphanidermatum is one of the most prevalent Pythium species in
greenhouses, and is more aggressive on poinsettia than the another prevalent species P.
irregularae.27 P. aphanidermatum isolates 106 and 319 were previously characterized for
sensitivity to etridiazole,2 and were selected from the culture collection of Dr. M.K.
Hausbeck at Michigan State University (MSU) and maintained on corn meal agar (CMA:
17 g/L corn meal agar). Prior to the study, the isolates were inoculated on poinsettia
stems and subsequently re-isolated from the diseased stems to ensure virulence.36
Zoospores of P. aphanidermatum were produced according to a previously established
method as described in SI.37 The concentration of initially produced zoospores was 1.7 Âą
0.9 × 104 zoospores/mL. The prepared suspension of the motile biflagellate zoospores
were equally split into five 500-mL capped bottles, and hand-shaken vigorously for 90
seconds to induce zoospore encystment. These suspensions of encysted zoospores were
then used later as the inoculum in irrigation water and for the poinsettias.
3.2.3. Plant and Irrigation Water.
One-month old cuttings of poinsettia (Euphorbia pulcherrima) Early Prestige Red
were obtained from a local commercial greenhouse. The cuttings were transplanted into
plastic nursery pots of 15 cm in diameter and 10.5 cm in height packed with a peat
potting mixture (Suremix, SunGro, Galesburg, MI). Fifteen plants were placed on each of
the six ebb-and-flow benches to ensure a sufficient number of replicates. In the second

78

experiment, the poinsettia cuttings were propagated from mature plants following a
commercial propagation recommendation38 and planted into the nursery pots as described
above. The poinsettia plants were approximately 6-week old at the start of each
experiment. A 20-5-19 (N-P-K) water-soluble fertilizer (JR Peters Inc, PA, USA) was
added to the irrigation water, and the initial nutrient concentration was 125 mg/L based
on nitrogen. Ground water (pH 7.85 Âą 0.22) was used as the source of irrigation water.
The pre-filter tank (or the holding tank in some filter-free treatments) in the irrigation
systems was filled with 120 L of the fertilized irrigation water. Plants were placed on the
benchtops with irrigation in operation for 2-days prior to inoculation to acclimate to the
soluble salts in solution. The suspension of encysted zoospores (490 mL of 1.7 × 104
zoospores/mL) was added to the pre-filter or holding tank and thoroughly agitated with a
wooden dowel. The resultant concentration of the encysted zoospores was 68 Âą 36
zoospores/mL. During the greenhouse experiments, the irrigation water was passed
through the filter unit and then stored in the holding tank immediately prior to an
irrigation event. Two additional inoculations were made two and four weeks after the
initiation of the experiment to increase disease pressure. To maintain a sufficient water
amount during each experiment, the fertilized irrigation water was added when the water
volume decreased to about 70–80% of the initial volume.
3.2.4. Greenhouse Experiments.
Two greenhouse experiments were conducted in the temperature-controlled
greenhouse under six experimental treatments, including: 1) a non-inoculated control
treatment without filtration and pathogen (−Control); 2) inoculated control treatment
without filtration or fungicide (+Control); 3) inoculated treatment with the sand filter; 4)

79

inoculated treatment with the AC filter; 5) inoculated treatment with the fungicide
etridiazole (Terrazole 35WP) application; and 6) ‘diseased plant’ treatment. In treatment
5, the etridiozole was applied at the labeled rate (250 mg/L) into the irrigation water in
the holding tank before the first inoculation to assess the effectiveness of the fungicide
treatment, in which no filter unit was used. In the “diseased plant” treatment (Treatment
6), 3 out of the 15 plants were directly inoculated with the Pythium zoospores, and then
placed randomly in the first experiment and at the back location near the drainage hole of
the bench in the second experiment, as shown in Figure 3.S3. In the “diseased plant”
treatment, Pythium zoospores were not introduced into the irrigation water, but were used
to inoculate the poinsettia plants. Briefly, 50 mL of the encysted zoospore suspension
was added to a 4-cm deep depression in the potting mix 2-cm from the stem of the
healthy plant. These pots were removed from the bench during inoculation to avoid
contamination of the bench, and then placed on the bench after the inoculation. This
treatment was designed to assess whether the pathogen could spread among the plants if
not directly introduced into the irrigation water. The poinsettia plants were irrigated twice
a day at 0900 and 1500 hr. The first experiment was initiated on October 29, 2014 and
concluded on January 6, 2015 (69 days in duration). The second experiment was initiated
on April 2, 2015 and concluded on June 19, 2015 (78 days in duration). The initiation day
was considered to be that of the first inoculation. Due to external streetlights providing
light contamination, the plants in the first experiment experienced an interruption of the
dark period that is required to initiate the flowering. Thus, in the second experiment, the
plants were covered with a thick black cloth at night to initiate flowering. The water
pressure inside of the filter unit was monitored in real time, along with the water

80

temperature in the holding tank, and air temperature and relative humidity in the
greenhouse, as detailed in SI. The irrigation water was sampled from the holding tanks at
the beginning, middle, and end of each experiment to determine the pH, electrical
conductivity (EC), and nutrient concentrations. After the pH and EC measurements, the
water samples were filtered through the 0.45-µm and stored in a −20 °C freezer for
nutrients analyses later by the MSU Soil and Plant Nutrient Laboratory.
3.2.5. Plant Assessments.
To evaluate the performance of the filtration systems in controlling Pythium root
rot outbreaks, the poinsettia plants were evaluated at the end of the experiment for foliar
and root biomass, root rot severity, and horticultural quality. The roots were evaluated for
root necrosis using a scale adapted from Boehm and Hoitink,39 where: 1 = no symptoms;
2 = mild root rot, <1/3 affected; 3 = intermediate root rot, 1/3 to 2/3 affected’ 4 = severe
root rot, >2/3 roots affected’ 5 = severe root and crown rot’ and 6 = dead plant. This
rating was made without removing the potting mix from the roots. In the second
experiment, the plants were rated for their horticultural quality38 based on the appearance
(e.g., color, height and bract area) on the scale from 1 (high aesthetic quality) to 5 (no
aesthetic quality) (Figures S4). At harvest, the roots were carefully washed and the fresh
biomass of the poinsettia shoots and roots were measured. The shoot and root samples
were then oven-dried at 60°C for 3 days and measured for their dry biomass. Isolation of
P. aphanidermatum from the roots of each plant was attempted to determine the root
infection ratio (IR) as described in SI.
Chlorophyll a, and b concentrations in the poinsettia leaves were measured using
the colorimetric method,40 as described in SI. To analyze the macro- and micro- nutrients

81

in the poinsettia leaves, the stems were removed and the remaining leaf tissues were
ground before being analyzed at the MSU Soil and Plant Nutrient Laboratory following
the standard methods.
3.2.6. Statistical Analysis.
Statistical analyses of the experimental data were performed with R software
using the “LSD” R package for parametric tests and the “coin” R package for a
nonparametric test such as the rating data (i.e., the ratings of root rot severity and
horticultural quality). Treatments were compared by one-way analysis of variance (p ≤
0.05). When a significant F value was determined, means were separated by the LSD’s
multiple comparison test. Also, the student’s t-test was used to compare paired samples.
Data of pre-inoculated plants in the ‘diseased plant’ treatment were not included in the
statistical analyses. To compare nonparametric data, such as root rot severity and
horticulture rating, the Kruskal-Wallis test were used with the “coin” R package. Its
statistical significance p-value was adjusted (p ≤ 0.034) by Bonferroni correction to
reduce the risk of committing Type I errors for multiple comparison.

3.3. RESULTS
3.3.1. Irrigation and Filtration Systems.
Six small-scale ebb-and-flow irrigation systems (Figure 3.S1) and filtration units
(Figure 3.S2) were used to evaluate the effectiveness of filtration and fungicide against P.
aphanidermatum and to demonstrate the pathogen dissemination among plants. The air
temperature and relative humidity were 26.8 ¹ 2.9 °C and 30 ¹ 7.4 % in the first
experiment, and 26.1 ¹ 3.3 °C and 36 ¹ 16.7 % in the second experiment, respectively

82

(Figure 3.S5). The water flow velocities through the AC and sand filters were 19.6 Âą 0.5
and 10.5 Âą 2.2 cm/min in the first experiment, and 18.6 Âą 1.3 and 8.6 Âą 1.0 cm/min in the
second experiment, respectively. Consequently, the water residence time in the AC and
sand filters was 2.5 Âą 0.1 and 4.9 Âą 0.9 minutes in the first experiment, and 2.6 Âą 0.3 and
6.3 Âą 0.4 minutes in the second experiment, respectively. The water flow velocity and
residence time were calculated based on three-day averages at the beginning of each
experiment (n = 6). Thus, the two experiments had consistent water velocities through the
filters. Operating water pressure of the AC and sand filters were maintained at 6.9 Âą 1.4
kPa (i.e., 1.0 Âą 0.2 psi) and 5.73 Âą 1.00 kPa (i.e., 0.83 Âą 0.15 psi), respectively (Figure
3.S6). Because filtration systems with a water velocity of 8.3–25 cm/min are classified as
rapid sand filtration,41-43 relative to slow sand filtration (0.17–0.5 cm/min), these filtration
systems are classified as fast-flow rate (8.6–19.6 cm/min) and low pressure. It was noted
that after about one month from the start of the second experiment, a significant water
flow reduction was observed, likely due to clogging by debris and biofilm.44 Flow
reduction due to clogging has also been observed with slow sand filters,31 and can often
be alleviated by backwashing.32 To maintain the proper water flow rate, backwashing was
conducted once a week after one month and then every other day during the last three
weeks in the second experiment. No significant reduction of water flow rate was found in
the first experiment so, backwashing was only performed a couple of times during the
final month of the experiment.
The pH and EC of the irrigation water were 7.8 Âą 0.4 and 1.6 Âą 0.3 mS,
respectively, during the experimental period (Figure 3.S7 and S8); water temperature was
in the range typical for a greenhouse (Figure 3.S9). The concentrations of macronutrients

83

(i.e., NO3−, P, K, Ca, Mg, and Na) in the irrigation water are shown in Table 3.S1. The
nutrient levels (i.e., NO3−, P, K, Ca, and Mg) in the AC filter treatment were generally
lower than those of other treatments, although the difference was less in the second
experiment. Thus, it appeared that the AC removed these nutrients from the irrigation
water45-49 and the final nutrient levels in the irrigation water were dependent on the
nutrient absorption by the poinsettia roots and the nutrient removal by the AC filter. It
appeared that the severe root rot in the AC filter treatment during the second experiment
(as shown later) may have caused insufficient nutrient absorption and subsequently
higher nutrient levels than those in the first experiment. Moreover, the micronutrients
(i.e., Fe, Cu, and Zn) were completely removed from the recycled irrigation water in the
AC filter treatment (Figure 3.S10). Thus, in both experiments there might be nutrient
deficiency for the plants in the AC filter treatment.
3.3.2. Horticultural rating.
The aesthetic quality of the poinsettias was assessed in the horticultural rating
scale from 1 to 5 at the end of the second experiment (Table 3.1 and Figure 3.1). The
plants in the first experiments did not properly develop the red color leaves because of
the interruption of the dark period by light contamination (Figure 3.S11), and thus could
not be evaluated for aesthetic quality. The inoculated control plants had an inferior
appearance with a horticultural rating of 3.0, and were not marketable. The plants
subjected to the AC filter treatment had the poorest aesthetic quality with a horticultural
rating of 4.7, whereas the plants in the remainder of the treatments received similar
horticultural ratings (1.4–2.3, Table 3.1).

84

3.3.3. Plant Assessment.
The inoculated control plants displayed severe root rot and stunting in both
experiments (Table 3.1, Figure 3.1, 2, S11, and S12). Root rot severity was visually
determined by assessing the percentage of root necrosis using a scale from 1 to 5 (Figure
3.S12 and Table 3.1). In the first experiment, the level of root necrosis in the inoculated
control plants (3.8 ± 1.0) was significantly (p < 0.0034) more than that of the noninoculated control (1.7 ± 1.0), sand filter (1.7 ± 0.9), AC filter (1.4 ± 0.5), and “diseased
plant” (1.8 ± 1.1) treatments. Root necrosis in the etridiazole treatment (2.5 ± 1.2) was
the second highest. In the second experiment, the levels of root necrosis in the inoculated
control plants (3.2 Âą 0.9), AC filter (3.2 Âą 0.6), and etridiazole (3.0 Âą 0.7) treatments were
significantly higher (p < 0.0034) than those of the non-inoculated control (1.1 Âą 0.4),
sand filter (1.2 ± 0.4) and ‘diseased plant’ (1.5 ± 1.2) treatments (Table 3.1). In particular,
the roots of plants in the AC filter treatment showed significant root necrosis (Figure
3.S12B) although P. aphanidermatum was not isolated from the roots. Presence of root
symptoms did not always correspond with isolation of P. aphanidermatum, perhaps due
to isolation inefficiency. In the first experiment, P. aphanidermatum was isolated from
the roots of 93, 80, and 50% of the plants for the inoculated control, etridiazole, and
“diseased plant” treatments, respectively, and the pathogen was not isolated from the
roots of the non-inoculated control, AC, and sand filter treatments (Figure 3.2). The
presence of P. aphanidermatum in the “disease plant” treatment did not result in
significant difference in the root necrosis compared with that of the non-inoculated
control treatment. It is likely due to the masking effect from other healthy plant roots
(Figure 3.S3). In the second experiment, P. aphanidermatum was isolated only from the

85

roots in the inoculated control (93% incidence). Significant root necrosis was observed
for some plants in the AC filter, “diseased plant”, and etridiazole treatments. It is possible
that rinsing the roots with water and surface-sterilizing them with 70% ethanol prior to
isolation prevented pathogen recovery. The root necrosis in the AC filter and etridiazole
treatments might result from nutrient deficiency and phytotoxicity respectively, whereas
it remained unexplained for the “diseased plant” treatment. It is important to note that
plants in the sand filter treatment consistently showed healthy roots similar to those of the
non-inoculated control, demonstrating the effectiveness of rapid sand filtration in limiting
Pythium infection.
Plant height and biomass were assessed to determine whether filtration effectively
maintained plant quality. In the first experiment, the sand filter, etridiazole, “diseased
plant”, and non-inoculated control treatments had significantly higher plant height, foliar
fresh weight, and root dry weight than those of the inoculated control plants (p < 0.05,
Figure 3.3). However, plants in the AC filter treatment had significantly lower fresh foliar
weight and root dry weight than those of the non-inoculated control (p < 0.05, Figure
3.3). In the second experiment, the sand filter, etridiazole, “diseased plant” and noninoculated control treatments had significantly higher plant height, and foliar fresh and
dry weight than those of the inoculated control and AC filter treatment (p < 0.05) (Figure
3.S12).
Chlorosis of young leaves was observed for plants in the AC filter treatment in
both experiments (Figure 3.S14). Chlorophyll a and b analysis for the medium-sized
leaves after harvest were significantly lower in the AC filter treatment than those of other
treatments (Table 3.2). The leaves had high levels of S and Ca and low levels of B, Zn,

86

Fe and Cu in the AC filter treatment, relative to the non-inoculated control. There was no
significant difference in the concentrations of N, P, K, Mg, Na, Mn, and Al in the leaves
from the AC filter treatment and the non-inoculated control treatments (Table 3.S2).

87

Table 3.1. Root rot severity and horticultural rating in the first and second experiments.
Exp. #

Parameter

−Control

+Control

AC filter

Sand filter

Diseased plant

Etridiazole

1st expt.

Root rot
severity

1.7 Âą 1.0 a*

3.8 Âą 1.0 b

1.4 Âą 0.5 a

1.7 Âą 0.9 a

1.8 Âą 1.1 a

2.5 Âą 1.2 ab

Root rot
severity

1.1 Âą 0.4 a

3.2 Âą 0.9 b

3.2 Âą 0.6 b

1.2 Âą 0.4 a

1.5 Âą 1.2 a

3.0 Âą 0.7 b

4.7 Âą 0.5 c

1.7 Âą 0.5 a

2.3 Âą 0.8 ab

1.9 Âą 0.6 a

2nd
expt.

Horticultural
1.4 Âą 0.6 a
3.0 Âą 0.8 b
rating
*Kruskal-Wallis with Bonferroni corrected, P < 0.0034

88

Table 3.2. Chlorophyll a and b in the first and second experiments.
−Control

+Control

AC filter

Sand filter

Diseased plant

Etridiazole

Chlorophyll a

42.71 bc*

49.09 a

16.48 d

39.16 c

47.93 a

46.12 ab

Chlorophyll b

76.28 bc

89.41 a

25.98 d

68.07 c

80.63 b

76.92 b

Chlorophyll a

16.2 ab

13.2 bc

4.0 d

13.0 c

15.1 abc

17.3 a

Chlorophyll b

110.4 ab

91.1 bc

25.4 f

88.6 c

101.9 abc

117.3 a

1st expt.

2nd expt.
*LSD test, P < 0.05

89

Figure 3.1. Poinsettias at the end of the second experiment (i.e., 78 days after inoculation).

90

Figure 3.2. Roots of the poinsettias at the end of the first experiment (i.e., 69 days after inoculation). IR: Infection ratio of
Pythium in roots.

91

Figure 3.3. Poinsettia height (A), foliar fresh biomass (B), foliar dry biomass (C), and root dry biomass (D) in the first
experiment (LSD test, P < 0.05).

92

3.4. DISCUSSION
3.4.1. Filtration Performance.
Our results demonstrated that the poinsettia plants under the fast-flow sand
filtration were consistently similar to the non-inoculated control, regarding the presence
of P. aphanidermatum, root necrosis, plant height and biomass, and horticultural quality.
Thus, the rapid sand filtration effectively limited P. aphanidermatum, likely by removing
P. aphanidermatum zoospores from the irrigation water. Many laboratory and greenhouse
experiments have consistently shown that sand filters can effectively remove pythiaceous
zoospores from water.35, 50-55 Similar results have also been found with the slow sand
filtration under experimental conditions.35, 53, 54, 56 The slow sand filtration was initially
developed for wastewater treatment through biological processes; a biological layer
termed “Schmutzdecke” is the most critical factor for purification.42 However, others
suggested that the main mechanisms to remove phythiaceous zoospores primarily rely on
physicochemical factors including surface attachment, pore straining, and adhesive
interactions of the zoospores in the porous media.50, 53, 54, 56 Thus, the physiochemicallycontrolled fast-flow filtration can be a viable alternative for treating recycled irrigation
water in the greenhouse. Slow sand filtration is often not suitable for greenhouse
production due to its low water flow rate and the large footprint of the filtration system.57
In this study, the flow rate of the rapid sand filter was about 40−50 times of that in typical
slow sand filters, and would thus would meet the water demand in commercial
greenhouses with a small footprint.
Nonetheless, a reduction was observed in the water flow rate in the sand filter
treatment over time, potentially due to clogging of the sand filter by debris or biofilm.44

93

The clogging can usually be easily remediated by backwash that is often performed in
typical filtration operations.32 Recently, Kim et al.

58

reported that a pungent oil of fresh

ginger (6 gingerol) reduced Pseudomonas aeruginosa biofilm formation up to 53% by
inhibiting quorum sensing-regulated virulence behaviors. In addition, several quorum
sensing inhibitors including RNAIII-inhibiting peptide, usnic acid, and a natural
secondary metabolite of lichen could also inhibit biofilm formation.59 However, no study
has been conducted on whether the quorum sensing inhibitors can maintain the water
flow rate in a sand filter. Thus, we used the backwash to maintain the water flow rate in
this study. Since performing the backwash sustained the desired water flow rate in our
sand filters, using rapid sand filtration may be an option to limit Pythium spp. in recycled
irrigation water in commercial greenhouses due to its low cost to install, maintain, and
operate.10
The AC filter effectively removed P. aphanidermatum zoospores, and no flow
reduction was observed during the two experiments. However, the biomass and height of
the plants under the AC filter treatment were, in general, significantly reduced compared
to the non-inoculated and sand filter treatments (Figure 3.3 and S13). Because P.
aphanidermatum was not detected in the relatively healthy-appearing roots in the AC
filter treatment of the first experiment, the leaf chlorosis and stunted growth may have
resulted from abiotic factors. As the AC removed essential micronutrients (e.g., Fe, Cu,
Mn, and Zn) from the irrigation water (Figure 3.S10), and the concentrations of Fe, Cu,
and B in the plant leaves (Table 3.S2) were in the deficient range,60, 61 the leaf chlorosis
might be due to micronutrient deficiency. The light green coloration of young leaves and
chlorosis indicate Fe deficiency.62,

63

Similarly, the kiwifruit showed the Fe deficient

94

symptom when the soil was amended with a wood-based biochar.64 Biochar produced by
pyrolysis characterized by carbon-rich, large surface area, high porosity, and lot of
functional groups similar to that of AC.65, 66 The removal of nutrients by the AC may
prevent its adoption for water treatments in commercial greenhouses.67 Despite its
effectiveness in removing the plant pathogen, the AC filters may not be ideal for the
commercial poinsettia production due to poor plant quality (Table 3.1 and Figure 3.1)
3.4.2. Fungicide application.
In the etridiazole treatment across both experiments, the poinsettias had similar
biomass and height to those in the non-inoculated control. However, root infection was
not prevented by the fungicide application. P. aphanidermatum was isolated from roots
exhibiting an intermediate level of root rot severity (Table 3.1 and Figure 3.2) in the first
experiment. In the second experiment, the plant roots also displayed an intermediate level
of root rot severity, but no P. aphanidermatum was isolated. When etridiazole was
incorporated into the growing medium, necrosis was reduced at the stem base of
cucumbers, but did not decrease recovery of P. aphanidermatum from the roots in an
ebb-and-flood floor system.6 In a hydroponic system, etridiazole applied in the
recirculating irrigation water reduced the root rot of Hedera spp., but not as effectively as
the fungicide mefenoxam.68 The presence of P. aphanidermatum in the poinsettia roots in
the first experiment suggested that the rate of etridiazole in the irrigation water may not
be adequate in controlling infection. Etridiazole did not cause 100% mortality to the
zoospores of sensitive isolates of P. aphanidermatum in vitro.2 Additionally, the
application of sub-lethal doses of fungicides may increase the resistance of Pythium spp.
to fungicides and exacerbate disease symptoms.69

95

Plants may not be adequately

protected if the fungicide concentration is below the threshold necessary to prevent
disease. Moreover, etridiazole may cause phytotoxicity in the poinsettia plants. Severe
root rot occurred with the etridiazole treated water in the absence of P. aphanidermatum
in the second experiment, suggesting that the recommended dosage of this fungicide may
cause phytotoxicity in the roots or induce root rot symptoms.70-72
3.4.3. Pathogen inter-plant transmission.
To demonstrate pathogen transmission from plant to plant, a study was designed
that randomly placed three inoculated poinsettia plants among 12 healthy plants. In the
first experiment, P. aphanidermatum from inoculated plants was transferred to healthy
plants. These infected plants did not show reduced biomass, but exhibited root rot
symptoms by the end of experiment (Figure 3.S3A). Pythium transmission from
inoculated to healthy plants was less efficient than infesting the irrigation water.14 The
pathogen did not spread from inoculated to healthy plants when inoculated plants were
placed at the back as shown in Figure 3.S3B. It is possible that the released zoospores
from the infected roots could be drained away with the irrigation water, and then become
attached to the surfaces of pipes and walls during the encystment.73 It is unlikely that the
zoospores would be transported against the flow direction as the motile zoospores can
only move up to a few millimeters to centimeters by active swimming.74-76 Thus, the
zoospores were possibly transported by irrigation water to infect the plants in the first
experiment, as the infected plants were located along the flow direction in the middle of
the bench (Figure 3.S3A).

96

3.5. IMPLICATIONS
In our study, P. aphanidermatum was effectively removed by the sand and AC
filters during fast-flow filtration with low water pressure. The rapid sand filter maintained
poinsettia quality compared to the non-inoculated control. The AC filter can also remove
the essential nutrients from the irrigation water, and cause Fe deficiency symptoms, thus
the use of an AC filter in the recirculating irrigation systems may not be preferred, unless
the nutrients can be applied separately. The application of etridiazole did not completely
prevent Pythium infection (e.g., root rots), but plant quality in terms of biomass, height
and horticultural quality was not compromised. Thus, fungicides may still be needed to
control the Pythium outbreak. When the poinsettia plants were randomly infected by P.
aphanidermatum, the pathogen spread among plants in the absence of any treatment,
suggesting the need of proactive measures to control the pathogen transmission either by
fungicide application or filtration.
In summary, our proof-of-concept study suggests that filtration of irrigation water
can effectively reduce crop disease outbreaks in greenhouses with ebb-and-flow and
flood-floor production systems. This could decrease the use of fungicides and promote
crop and environmental health. Future work should focus on assessing the longevity of
the system performance by optimizing filter media and operation parameters. For
instance, the filter design can be improved by incorporating anti-clogging mechanisms
such as deeper coarse layer at the inlet, or removable screening for dislodging
accumulated debris. Sand grain size could also be optimized to improve water flow,
while maintaining the zoospore removal efficiency. When using the AC filter, the
nutrients should be applied via ways other than irrigation water. Finally, the irrigation

97

frequency and duration, and backwashing scheduling may be optimized to ensure the
continuous system performance.

3.6. ACKNOWLEDGMENTS
This research was partly supported by American Floral Endowment, and
Michigan State University AgBioResearch. The views and opinions of the authors
expressed herein do not necessarily reflect those of funding organizations and Michigan
State University. Mention of tradenames or commercial products does not constitute
endorsement or recommendation for use.

98

APPENDIX

99

APPENDIX
SUPPORTING INFORMATION
S1. Supplemental Materials and Methods
S1.1 Construction of Ebb-and-Flow Irrigation Systems
To test the effectiveness of filtration units in controlling disease outbreaks in
greenhouse-grown poinsettias, six self-contained ebb-and-flow irrigation systems
(including optional filtration units) were constructed (Figure 3.S1Error! Reference
source not found.). A typical irrigation system consisted of an 2.4 m × 1.2 m black
plastic ebb-and-flow bench (Hummert, St. Louis), an optional filtration unit, two 130-L
holding tanks, two 12V-centrifugal water pumps, two check valves, two auto valves, two
water-level sensors, and a timer. The irrigation water was withdrew from the pre-filter
tank by one water pump, passed through a check valve, the filter unit, an auto valve (i.e.,
1/2-inch motorized ball valve, Model number: MV-2-20-12V-R01-1, Misol, China), and
then stored in the holding tank until a pre-scheduled irrigation time controlled by a timer.
At the time of irrigation, the irrigation water in the holding tank was pumped into the
ebb-and-flow bench via a check valve until reaching a desired watering height (i.e., 3–4
cm or 10 mins of pumping time). The two check valves were installed to prevent the
backflow. One check valve was next to the pump connected to the pre-filter tank, and the
other next to the pump connected to the holding tank (Figure 3.S1). The irrigation water
in the bench was kept for a desired irrigation period before being drained back to the prefilter tank by opening an auto value (i.e., the 3/4-inch motorized ball valve). Two
magnetic float water-level sensors (Model number: a11062100ux0008, Uxcell, VA,
USA) were installed in the pre-filter tank and holding tank, respectively. The water-level

100

sensor in the pre-filter tank detects the irrigation water drained from the bench, and then
the water pump is automatically turned on to deliver the water to the inlet of the filter
unit. The second water-level sensor turns off the water pump connected to the holding
tank, if the water level reaches the minimum level so as to prevent air entry into the
pump.
The filter unit design is described in detail next. Activated carbon (AC) and sand
were used as filter media. Operating water pressure of the AC and sand filters was
maintained at 6.9 Âą 1.4 kPa (i.e., 1.0 Âą 0.2 psi) and 5.73 Âą 1.00 kPa (i.e., 0.83 Âą 0.15 psi),
respectively. The water pressure was measured by a pressure transducer with a range of
0–15 psi (Model: MK-15, China) at the top of the filters and then recorded in a datalogger (Model: MCR-4V; TandD, Japan). All of the irrigation systems were sterilized
before each experiment. Specifically, a solution of > 30% of household bleach (i.e.,
6.15% NaClO solution) was applied in the benches and tanks using sprayers, and any
adhering grime or algae was removed using scrub brushes. Then, 10 L of a 5% bleach
solution was added to the pre-filter tank and allowed to recirculate a couple of times in
the absence of a filter unit. After the cleaning, the systems were thoroughly rinsed several
times with tap water and air-dried for several days.
S1.2. Filter Unit Design
Low-pressure sand and AC filters were constructed for the greenhouse
experiments (Figure 3.S2). Each filter unit was made with a PVC pipe of 50 cm in length
and 15.2 cm (6 inches) in diameter. The bottom of each filter column was sealed with an
end cap fitting and the top of each filter was assembled with a coupling, an adapter
fitting, and a plug fitting in order. Two types of filter media, i.e., sand (99.69% silica,

101

GranusilÂŽ ) and AC (Filtrasorb 300, CalgonCarbon, USA), were used. The particle size
distribution of the sand was 5.1% of 297-420 µm, 57.2% of 420-595 µm, 36.1% of 595841 µm, and 1.2 % of ≥ 841 µm. The effective size of AC particles was 0.8-1.0 mm. A 3cm layer of coarser sand (500–841 µm) was placed at the bottommost and upmost in the
sand filter to filter out large debris and thus minimize the clogging, but only at the bottom
of the AC filter. The total depth of filter media was 50 cm including the coarser sand
layers. All of filter media were used directly without washing. To support the filter media
and allow for the free drainage of the filtered water, two screens with different opening
sizes (i.e., 12.7 mm × 12.7 mm and 6.35 mm × 6.35 mm) were prepared and bent to be
fixed onto about the 2-cm length of the 15.2-cm PVC pipe using 12 screws and then
mounted inside the end of the bottom cap. A stainless screen with 100 × 100 µm opening
size was placed on the screens. The top of the filter media was also covered with the 100
× 100 µm and 12.7 × 12.7 mm screens in the similar manner as that at the bottom part to
filter large debris and allow for an even distribution of water flow (Figure 3.S2). The
bottom end cap was drilled and fitted with a 1/2-inch polypropylene bulkhead tank fitting
(TF050, Banjo, USA) to connect the outlet pipe along with a union fitting (Mueller/B
&K, USA) and a 1/2-inch motorized ball valve (Misol, China). The motorized ball valve
was open during the operation of the pump connected to the pre-filter tank. The pressure
sensors were installed at the inlet of each filter. All of the components were assembled
and sealed to make watertight columns.

102

Figure 3.S1. Schematic of the ebb-and-flow irrigation system constructed in the greenhouse.

103

Figure 3.S2. Schematic of filter unit (a. 12-V water pump, b. check valve, c. union fitting,
d: pressure sensor, e. PVC plug fitting, f. PVC adapter fitting, g. PVC coupling, h. PVC
end cap fitting, i. Bulkhead fitting, and j. motorized ball valve).

104

S1.3. Preparation of Pythium Zoospores
The Pythium isolates were grown on the V8-agar culture for 5 days. The V8-agar
culture was then divided into six strips and separated into two sterile petri dishes of 100
mm in diameter. The petri dishes were flooded with sterile distilled water (SDW),
incubated at 30 °C for 24 h, drained, rinsed, and flooded with another 25 mL of SDW.
After incubation for 10 h at ambient temperature (21 ¹ 2°C), the zoospores were released
from the sporangia, and then were transferred to a 2-L beaker half filled with SDW. To
determine the concentration of the zoospore suspension, a 1-mL aliquot was placed into a
1.7 mL microcentrifuge tube, vortexed for 70 seconds to induce the zoospore encystment,
and then a 10 ÂľL aliquot was pipetted onto a clean hemocytometer for counting (BrightLine, Hausser Scientific, Horsham, PA).

Figure 3.S3. Location of pre-inoculated plants (red), infected plant (yellow), and healthy
plants (green) at the end of the first (A) and second (B) experiments. The number in the
column is the root rot severity. The infection of the plants was identified by the isolation
of Pythium aphanidermatum.

105

S1.4. Pythium Isolation
To isolate P. aphanidermatum from the poinsettia roots, the root mass was rinsed gently under running tap water to
remove any adhering potting mixture. Three water-soaked or discolored roots were selected per plant and were surfacesterilized in 70% ethanol, blotted dry, and plated onto amended-CMA and incubated at 30°C for 24 h. Pythium colonies were
transferred to CMA and confirmed as P. aphanidermatum by sporangial and oospore morphology.77 The number of plants with
the presence of P. aphanidermatum was divided by the total number of plants (n = 15) to determine the root infection ratio
(IR).

Horticultural rating #1

Horticultural rating #2

Horticultural rating #3

Horticultural rating #4

Horticultural rating #5

Figure 3.S4. Horticultural rating scale in the second experiment (#1 = high aesthetic quality, and #5 = no aesthetic value).

106

S1.5. Chlorophyll Analysis
Chlorophyll a, and b concentrations in the poinsettia leaves were measured using
a colorimetric method,40 as described at the following. Three poinsettias were randomly
selected and 0.5 g of the leaf sample was collected from more than 3 leaves in the
middle-part of each plant. The fresh leaf samples were processed immediately after
collection. The weighted leaf samples were homogenized with the addition of 10 mL of
80 % acetone. The extract was centrifuged at 2500 rpm for 5 min, and the supernatant
was diluted 10 times by adding 80% acetone. The final extracted solution was analyzed
for the chlorophyll concentrations by a spectrophotometer (Varian Cary 50 Bio,
McKinley, New York).

107

Figure 3.S5. Air temperature and relative humidity in the first (A) and second (B)
experiments.

108

A

0

10

20

30

40

50

60

70

Date
Days

B

0

10

20

30

40

50

60

70

Date
Days

Figure 3.S6. Water pressure in the activated carbon (AC) and sand filters in the first (A)
and second (B) experiments.

109

Figure 3.S7. pH of irrigation water measured in the first (A) and second (B) experiments.

110

Figure 3.S8. Electrical conductivity (EC) of irrigation water measured in the first (A) and
second (B) experiments.

111

Figure 3.S9. Irrigation water temperature in the holding tank for the first (A) and second
experiments (B).

112

Table 3.S1. Results of nutrients in the irrigation water during the first and second experiments.
Treatments
NO3−
P
K
Ca
Days*
0
22 69
0
22 69
0
22
69 0
22 69 0
−Control
92 128 172 7.7 9.7 8.8 111 117 135 95 123 150 38
+Control
88 128 263 7.5 6.8 8.8 100 109 149 95 123 177 41
1st Sand
91 117 150 6.2 7 8.6 91 96 109 95 123 136 35
AC
41 53 95 2.6 2.7 2.4 100 91
99 55
95 109 36
Etridiazole
83 88 147 6.2 6.6 5.3 94 110 126 95 109 150 39
Diseased plant
85 116 151 6.6 5.6 14.5 94 107 110 95 123 123 36
Ground water
0 n.a. n.a. 0.4 n.a. n.a. 2 n.a. n.a. 109 n.a. n.a. 36
Days
5
38 78
5
38 78
5
38
78 5
38 78 5
−Control
87 154 154 5.5 6.2 6.4 96 122 125 123 150 150 44
+Control
89 154 150 7.7 9.2 5.2 103 132 143 123 150 164 41
Sand
62 100 63
4 3.5 2.3 91 97
54 123 123 109 42
2nd
AC
36 72 94 1.8 2.2 2.6 94 109 111 68 123 136 41
Etridiazole
83 98 80 6.4 2.8 4.4 103 123 100 123 109 109 45
Diseased plant
88 163 163 7 4.7 3.7 106 147 147 123 164 164 45
Ground water
0 n.a. n.a. 0.1 n.a. n.a. 2 n.a. n.a. 95 n.a. n.a. 35
* The numbers in the “days” means days calculating from the start of the experiments.

113

Mg
22 69
47 59
60 73
45 53
35 44
45 83
48 51
n.a n.a.
38 78
56 57
53 62
48 44
51 54
54 47
62 63
n.a. n.a.

0
16
16
16
16
21
16
17
5
26
26
25
25
31
25
15

Na
22
21
22
21
18
24
20
n.a.
38
30
30
26
29
36
31
n.a.

69
29
38
26
22
35
23
n.a.
78
24
27
19
27
27
28
n.a.

0.35
0.3

AC

A

0.14

-Control

0.1

0.2

0.08

0.15

0.06

0.1

0.04

0.05

0.02

0

0

0.2 4/3

4/23

5/13

6/2
AC

C

6/22

0.8 4/3

-Control

Zn (ppm)

0.1

0.05

4/23

5/13

6/2
AC

D

0.7
0.6

0.15
Mn (ppm)

-Control

Cu (ppm)

Fe (ppm)

0.25

AC

B

0.12

6/22
-Control

0.5
0.4
0.3
0.2

0.1
0

0
4/3

4/23

5/13

6/2

6/22

4/3

4/23

5/13

6/2

6/22

0

20

40

60

80

0

20

40

60

80

Date
Days

Figure 3.S10. Micronutrient concentrations in the irrigation water in the activated carbon (AC) and non-inoculated control
treatments during the second experiment.

114

Figure 3.S11. Poinsettias at the end of the first experiment (i.e., 69 days after inoculation).

115

Figure 3.S12. Poinsettia roots for evaluating root necrosis in the first (A) and second (B)
experiments. White color of the roots indicates a healthy root system, and dark color and
dispersed potting soil suggest a rot root system. The number is the rating of root rot
severity (1 = no symptoms, 2 = mild root rot, <1/3 affected, 3 = intermediate root rot, 1/3
to 2/3 affected, 4 = severe root rot, >2/3 roots affected, 5 = severe root and crown rot, and
6 = dead plant).
116

Figure 3.S13. Poinsettia height (A), foliar fresh biomass (B), and foliar dry biomass (C) in the second experiment (LSD test, P
< 0.05).

117

Table 3.S2. Micronutrients in the leaves of the poinsettias in the second experiment.

AC
−Control

N

S

P

3.07 Âą 0.21 a*
3.39 Âą 0.02 a
B

0.33 Âą 0.01 a
0.29 Âą 0.01 b
Zn

0.52 Âą 0.12 a
0.41 Âą 0.02 a

K
%
4.52 Âą 0.51 a
3.57 Âą 0.04 a
Mn

Mg

Ca

Na

0.97 Âą 0.19 a
0.68 Âą 0.06 a
Fe

2.04 Âą 0.17 a
1.55 Âą 0.03 b
Cu

0.030 Âą 0.014 a
0.013 Âą 0.005 a
Al

mg/kg
AC
−Control

14.7 Âą 4.7 a
41.7 Âą 2.9 b

31.3 Âą 4.2 a
59.3 Âą 3.3 b

129.7Âą16.7 a
95.7Âą7.6 a

44.0Âą8.3 a
74.0Âą3.3 b

*t-test, P < 0.05

118

2.0Âą0.8 a
5.0Âą0.8 b

41.7Âą13.3 a
39.3Âą5.4 a

Figure 3.S14. Color of leaves comparison between non-inoculated control and AC filter
treatments in the first (A) and second (B) experiments.

119

S1. Poinsettia Greenhouse Experiments Raw Data
Table 3.S3. Experiment A: Poinsettia Foliar fresh weight (g)
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

- Control
219
198
188
185
201
186
191
193
118
165
151
204
187
96
90
171.5
38.56

+ Control
88
103
157
125
136
102
110
132
112
96
114
94
77
118
105
111.3
19.69

AC filter
159
150
149
145
177
115
133
165
161
135
138
184
149
121
170
150.1
19.08

c

Sand filter
173
179
195
164
115
200
147
216
146
195
188
178
166
178
182
174.8
24.2

Diseased
plant
138
246
I.P c
I.P
152
186
175
169
223
176
180
215
183
214
I.P
188.1
29.66

Etridiazole
160
123
197
171
202
131
156
174
226
196
176
207
173
163
156
174.1
27.10

I.F = pre-infected plant = biomass of initially infected plant was not included for data
analysis.
d
Plants were cut and used as propagation for second experiment after fresh weight
measurement.

120

Table 3.S4. Experiment A: Poinsettia foliar dry weight (g)
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

26.5
23.7
25.3
22.2
25.5
N.Ad
23.2
N.A
16.4
20.1
N.A
25.9
27.9
N.A
13.0
22.7
4.3

12.5
14.0
20.4
16.5
17.2
13.6
15.3
18.5
15.1
13.1
14.4
13.0
10.0
15.4
13.4
14.8
2.5

20.8
18.3
18.2
N.A
21.2
13.8
16.1
20.0
19.6
N.A
16.8
23.0
18.1
15.0
19.4
18.5
2.5

N.A
22.1
24.3
19.8
15.0
25.9
17.3
27.8
N.A
24.8
23.8
22.8
N.A
23.1
24.6
22.6
3.5

Diseased
plant
16.9
30.0
I.P
I.P
20.7
N.A
N.A
N.A
27.0
21.6
23.7
26.0
22.7
26.0
I.P
23.8
3.7

Etridiazole
20.6
16.6
N.A
22.0
25.7
16.8
20.5
N.A
29.2
25.1
N.A
26.4
21.8
21.8
20.9
22.3
3.6

Table 3.S5. Experiment A: Poinsettia root dry weight (g)
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

5.6
5.6
7.0
4.6
6.2
4.1
5.0
7.1
3.3
3.2
3.8
4.9
4.9
2.8
2.0
4.7
1.4

2.5
2.9
3.8
2.8
3.2
1.8
2.8
3.5
2.3
2.8
2.5
2.5
1.4
2.3
2.9
2.7
0.6

3.3
2.9
3.3
3.9
2.9
3.5
2.2
3.3
4.2
3.2
3.0
3.8
3.9
2.7
2.7
3.2
0.5

5.9
4.5
7.7
3.6
2.5
4.8
3.6
3.1
4.3
5.4
6.0
6.0
2.8
4.2
7.5
4.8
1.6

121

Diseased
plant
3.4
6.5
I.P
I.P
4.7
5.6
5.0
4.7
4.6
4.7
4.6
5.1
5.4
5.7
I.P
5.0
0.7

Etridiazole
4.1
2.8
6.3
3.9
4.6
6.1
3.5
5.1
6.9
6.1
3.5
6.1
5.7
3.9
4.7
4.9
1.2

Table 3.S6. Experiment A: Poinsettia height (cm)
Number

- Control

+ Control

AC filter

Sand filter

Diseased
plant

Etridiazole

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

35
38
35.5
33
34
34
32
35.5
29
31
33
33
35.5
30
24

25
27
32
26
33
24
27
28
27
26.5
24.5
24
19
25.5
27

31
28
27.5
28.5
30
31
25.5
29.5
35
31
33
35
34
29.5
32

31.5
31
36
31
32
38
35
38.5
31
36.5
38
37
34
33.5
40

27.5
36.5
I.P
I.P
31.5
33
29.5
32
32.5
34.5
35
36
37
35
I.P

30
28.5
34
36
39.5
33
27
33
36
38
36
36.5
32.5
34
33

Mean
SD

32.8
3.3

26.4
3.2

30.7
2.7

34.9
3.0

33.3
2.8

33.8
3.3

Table 3.S7. Experiment A: Poinsettia root rot severity
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

1
1
2
1
1
1
1
1
4
1
3
1
2
2
3
1.7
0.94

5
5
2
3
5
4
4
4
5
3
4
4
4
2
3
3.8
0.98

1
1
2
1
1
2
2
1
2
1
1
2
2
1
1
1.4
0.49

1
1
1
1
3
2
1
4
2
2
1
1
2
1
2
1.7
0.87

122

Diseased
plant
1
1
I.P
I.P
4
2
2
1
2
1
4
1
1
2
I.P
1.8
1.07

Etridiazole
4
5
2
3
2
3
3
3
1
1
3
1
1
3
3
2.5
1.15

Table 3.S8. Experiment A: Poinsettia chlorophyll a and b

Chlorophyll
a
Chlorophyll
b

- Control

+ Control

AC filter

Sand filter

Diseased
plant

Etridiazole

41.5
42.5
44.1
71.5
77.7
79.7

51.0
47.5
48.8
95.5
87.9
84.8

18.1
16.4
15.0
28.7
26.1
23.2

38.2
43.3
35.9
67.0
76.3
60.9

48.2
47.6
47.9
76.9
82.9
82.1

46.1
43.7
48.6
74.2
74.8
81.8

continue

123

Table 3.S9. Experiment B: Poinsettia foliar fresh weight (g)
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

203
156
277
201
153
252
208
145
71
279
133
159
249
240
140
191.1
58.50

120
124
221
35
47
51
176
54
155
189
97
68
147
214
96
119.6
60.21

135
47
54
108
82
93
36
41
107
56
159
96
76
89
85
84.3
33.52

179
184
157
182
170
147
259
172
117
207
119
204
244
141
129
174.1
40.60

Diseased
plant
224
145
223
203
186
175
34
172
185
162
130
226
I.P
I.P
I.P
172.1
50.98

Etridiazole
182
176
160
247
204
148
199
227
166
125
135
139
131
165
198
173.5
34.94

Table 3.S10. Experiment B: Poinsettia foliar dry weight (g)
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

35.1
28.2
48.3
33.4
24.8
45.0
37.3
25.5
12.4
46.9
21.9
26.8
42.5
43.5
25.0
33.1
10.31

22.8
22.5
42.3
6.2
19.0
9.6
31.6
10.3
26.6
34.4
17.1
12.1
26.4
36.9
18.0
22.4
10.35

19.4
6.5
8.0
15.2
11.3
14.8
4.2
6.3
17.1
8.6
26.1
13.9
11.1
12.4
14.1
12.6
5.49

32.5
30.9
25.6
31.6
27.9
25.9
44.5
29.2
19.5
34.1
20.3
32.6
41.8
22.0
21.2
29.3
7.13

124

Diseased
plant
41.2
24.8
41.9
36.1
33.0
33.5
7.7
32.8
33.1
30.9
24.2
42.9
I.P
I.P
I.P
31.8
9.24

Etridiazole
31.5
32.1
27.3
45.3
34.6
25.2
33.7
39.6
28.5
21.2
22.6
24.1
22.1
27.6
34.7
30.0
6.62

Table 3.S11. Experiment B: Poinsettia height (cm)
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

36
29
35
40
32
31
38
28
25
39
34
36
37
41
28
33.9
4.73

22
20.5
31
21
22
23
32.5
22.5
29
32
26
22
27
40
25
26.4
5.37

26.5
23
18
27.5
23
26
22
21
25
23
36
24.5
31
20.5
21
24.5
4.36

34
36
32
33
30
35
39
33
32
31.5
27
32
30.5
30
31
32.4
2.76

Diseased
plant
33.5
35
37
34
31
38
23
35
30
28
35
38.5
I.P
I.P
I.P
29.7
8.19

Etridiazole
31.5
33
32
45
33
30.5
36
44
45
26
35
35
37
38
32
35.5
5.36

Table 3.S12. Experiment B: Poinsettia root rot severity
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15

1
1
1
1
1
1
1
1
2
1
1
1
1
1
2

4
3
3
4
4
4
3
3
2
4
2
2
4
2
4

3
4
3
3
3
3
4
4
4
3
3
3
3
2
3

2
1
1
1
1
1
1
1
2
1
1
1
1
1
2

Diseased
plant
1
1
1
1
2
1
5
1
1
1
2
1
I.P
I.P
I.P

1.1
0.34

3.2
0.83

3.2
0.54

1.2
0.40

1.5
1.12

Mean
SD

125

Etridiazole
2
3
2
3
3
2
3
3
3
3
4
4
3
4
3
3.0
0.63

Table 3.S13. Experiment B: Poinsettia horticultural rank
Number

- Control

+ Control

AC filter

Sand filter

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

1
2
1
1
1
1
2
2
2
1
1
1
1
1
3
1.4
0.61

3
3
2
4
4
4
3
3
2
2
3
4
3
2
3
3.0
0.73

5
5
5
5
5
4
5
4
5
5
4
5
5
4
5
4.7
0.44

2
1
2
2
2
2
1
1
2
2
2
2
2
2
1
1.7
0.44

Diseased
plant
3
2
2
3
2
2
4
2
1
2
2
2
I.P
I.P
I.P
2.3
0.72

Etridiazole
2
2
2
2
2
1
2
2
1
3
2
2
3
2
1
1.9
0.57

Table 3.S14. Experiment B: Poinsettia chlorophyll a and b (mg/ml)
- Control

+ Control

AC filter

Sand filter

Diseased
plant

Etridiazole

Chlorophyll
a

14.1
17.6
17.0

11.5
15.3
12.7

1.0
5.1
6.1

13.0
14.2
11.7

16.5
14.9
13.8

17.3
16.4
18.3

Chlorophyll
b

98.7
120.4
112.1

80.5
102.6
90.1

6.8
32.4
36.9

87.8
96.7
81.3

112.2
101.3
92.1

116.9
110.4
124.5

continue

126

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127

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Ufer, T., Werres, S. K., Posner, M., and Wessels, H.-P., Filtration to eliminate
Phytoph-thora spp. from recirculating water systems in commercial nurseries.
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minimise the risks of spreading root-infecting pathogens in closed hydroponic
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Climates: Current Trends for Suistainable Technologies 559, 2000; 2000; pp 495502.

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Wohanka, W.; Helle, M., Suitability of various filter media for slow filtration.
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Kim, H.-S.; Lee, S.-H.; Byun, Y.; Park, H.-D., 6-Gingerol reduces Pseudomonas
aeruginosa biofilm formation and virulence via quorum sensing inhibition.
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Algburi, A.; Comito, N.; Kashtanov, D.; Dicks, L. M.; Chikindas, M. L., Control of
biofilm formation: antibiotics and beyond. Applied and environmental microbiology
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Campbell, C., Reference sufficiency ranges for plant analysis in the southern region
of the United States. 2000.

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Hochmuth, G.; Maynard, D.; Vavrina, C.; Hanlon, E.; Simonne, E., Plant tissue
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GarzĂłn, C. D.; Molineros, J. E.; YĂĄnez, J. M.; Flores, F. J.; del Mar JimĂŠnez-Gasco,
M.; Moorman, G. W., Sublethal doses of mefenoxam enhance pythium damping-off
of geranium. Plant Dis. 2011, 95, (10), 1233-1238.

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thiophanate-methyl mixture to control Fusarium root disease of container-grown
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134

CHAPTER 4

CONTROL OF PHYTOPHTHORA CAPSICI DISEASES IN GREENHOUSE
SQUASH BY FAST-FLOW FILTRATION

ABSTRACT
Phytophthora spp. are challenging to manage in greenhouse production of
ornamentals and vegetables when recirculating irrigation water is used. The objective of
this study was to investigate the effectiveness of fast-flow filtration in limiting
Phytophthora diseases in a model greenhouse vegetable system. Two greenhouse
experiments were conducted to test the effect of filter media type (i.e., sand and iron
oxide coated media [IOCM]), and application of the fungicide etridiazole in controlling
P. capsici root and crown rot of squash. Fast-flow filtration through the IOCM filter was
more effective in limiting P. capsici than the sand filter. The plants in the IOCM filter
and fungicide treatments did not display disease symptoms, whereas 44–100% of the
plants in the sand filter treatment exhibited disease symptoms including wilting and
stunting. Squash plants in the IOCM treatment displayed chlorosis of older leaves and
slight stunting symptoms due to nutrient deficiency. Plants treated with fungicide
developed phytotoxicity symptoms that included interveinal chlorosis. In summary, plant
pathogens such as Phytophthora spp. can be removed from irrigation water using fastflow filtration through the IOCM and this filtration method may be a promising
alternative for disease management in vegetable production greenhouses.

135

4.1. INTRODUCTION
Greenhouses provide optimal conditions to produce vegetables of high quality and
yield in a small space. U.S. greenhouse vegetable production increased at a rate of 25%
per year from 2002–2006 1. Relatively small area of greenhouse production provides a
significant amount of production of the total agricultural production value 2. Pest
management is critical to greenhouse vegetable production

3

. The oomycete,

Phytophthora capsici, is considered one of the most destructive plant pathogens, causing
seedling damping-off, stunting, and stem, crown, and root rot in many vegetable crops.
This results in limiting production and causing devastating crop losses

4-7

. Phytophthora

and Pythium spp. may be introduced to the greenhouse via infected seedling plug plants
or soil and dust

8, 9

. Once introduced, the pathogen can survive on plant containers,

benches, and walkways 10. Hong and Moorman 8 reported that 16 Phytophthora spp. and
26 Pythium spp. were isolated from nursery and greenhouse operations. Control of
Phytophthora spp. can be especially challenging because plants can be infected but do
not show symptoms until the disease is too advanced for treatment 11.
Recycling irrigation water in the greenhouse offers environmental and economic
benefits but presents unique challenges due to the potential of disseminating water mold
pathogens such as Phytophthora 8. Recycling irrigation water in greenhouses is becoming
increasingly common. Meador, et al.

12

reported that 12 of the 24 surveyed greenhouses

in the U.S. recycled their irrigation water; the largest daily water use was as high as
1,000,000 gallons. In their study, the three largest water users recycled 75 to 100% of
their daily irrigation water. Phytophthora spp. release motile zoospores into the flowing
water with pathogen transmission exacerbated by irrigation water recycling

136

13, 14

. These

pathogens become established under the warm and wet conditions of the greenhouse
environment. P. capsici-resistant cultivars are not currently available, and the pathogen’s
dormant spores (oospores) can survive without a host for many years

15

. More

importantly, the cycle of plant infection and sporangia and zoospore production is rapid
16, 17

, therefore, even low levels of Phytophthora spp. in recycled irrigation water can

result in an epidemic if effective water treatment is not implemented 18.
Various chemical and physical methods have been proposed to disinfect the
contaminated recirculation irrigation water in greenhouses, including filtration,
chlorination, copper ionization, ozonation, UV light, activated peroxygens, chlorine
dioxide, and heat 8, 19. Many of these treatments are costly. Use of effective fungicides is
limited due to a lack of products registered for use against Phytophthora spp. on
vegetables produced in the greenhouse. Development of pathogen resistance to
fungicides is also a primary concern with Phytophthora spp.

16, 20

resulting in a lack of

fungicide efficacy and control failure (Lamour and Hausbeck, 2003; Lu et al., 2011).
Moreover, phytotoxicity to some crops may occur when high chemical rates are applied
21

. Water filtration is an cost-effective technique to remove oomycete pathogens such as

Phytophthora and Pythium spp. 8, 22, 23. Currently, screen or disk filters with a pore size >
100 Îźm are used to remove large particles such as potting soil particles and plant debris
to avoid clogging of the irrigation drippers

19, 24

. These filters are also used as pre-

treatment for other disinfection methods such as heat, ozone or UV radiation treatment 19.
Membrane filters of relatively small pore size (< 5Âľm, i.e., microfiltration) can be used to
remove oomycete pathogens. However, microfiltration often requires relatively high
water pressure and costly maintenance due to clogging and leaking problems

137

25

. Slow

sand filtration has been used in drinking water treatment, and also studied for removal of
plant pathogens in greenhouse applications
been adopted in commercial greenhouses

21

22, 26-28

. However, sand filtration has rarely

. Slow sand filters are easy to operate and

maintain, but water flow is often low at a range of 100–300 L/m2/h

21, 25

. The low water

flow rate can be a limiting factor to the wider adoption of slow sand filtration in the
greenhouses. Additionally, the performance of sand filtration for controlling the plant
pathogens is variable

23, 29

. Therefore, more research, particularly on fast-flow filtration,

is needed to improve the performance and adoption of filtration for plant pathogen
control in the greenhouse.
Removal of Phytophthora spp. from irrigation water by depth-bed filter filtration
depends on pathogen surface properties (e.g., surface charge, and bio-adhesive) and
physicochemical characteristics of filter media such as grain size and surface charge 28-31.
A previous study found that most encysted zoospores of P. capsici can be removed from
recirculating irrigation water, as a result of strong surface attachment, pore straining, and
adhesive surface interactions

30

. Iron-oxide coatings significantly increased the removal

of P. capsici encysted zoospores 30.
The objective of this study was to investigate the effectiveness of fast-flow
filtration of recycled irrigation water to limit disease caused by P. capsici in a model
greenhouse vegetable production system. Six small-scale recirculating irrigation systems
simulated ebb-and-flow and flood-floor systems that were constructed to simultaneously
test the effect of filter media type (i.e., sand and iron oxide coated media [IOCM]), and
fungicide application on controlling P. capsici. Squash was used as a model crop because
the plant is very susceptible to P. capsici and symptoms develop quickly 32.

138

4.2. MATERIALS AND METHODS
4.2.1. Irrigation and Filtration Systems.
Six small-scale irrigation systems were constructed as described

33

and used in

this study. Briefly, each irrigation system consisted of an ebb-and-flow bench connected
to an optional filtration unit, a 130-L holding tank, and an optional 130-L pre-filter tank.
Water flow was delivered by two hydraulic pumps located in the pre-filter and post-filter
holding tanks (Figure 4.S1). The system allowed the irrigation water to be pumped into
the ebb-and-flow bench, and then be drained back to the holding tank automatically at a
predetermined irrigation schedule controlled by timers. When a filter unit was installed,
the irrigation water was also passed through the filter unit (Figure 4.S1). The design of
the filter unit was described in detail elsewhere

33

. The filter was primarily made of

capped 6-inch PVC, and packed with either sand (99.69% silica, GranusilÂŽ ) or granular
IOCM. The particle size distribution of the sand was 5.1% of 297-420 µm, 57.2% of 420595 µm, 36.1% of 595-841 µm, and 1.2 % of ≥ 841 µm. The IOCM was provided by
MetaMateria Technologies (Columbus, OH), and manufactured by a proprietary process.
A monolith of IOCM was sealed in a fiberglass column. The IOCM is a highly porous
ceramic media produced by mixing and curing proper amounts of aluminates, silicates,
iron filer, surfactants, and gas forming agents without heating, and then surface-modified
with nano-sized iron oxide, resulting a specific surface area of about 100 m2/g. Its high
porosity and interconnected pores contribute to a high water permeability and,
subsequently, a fast water flow through the media at a low water pressure. The IOCM
was initially developed to sorb phosphorus from water 34, and was tested for the removal
of P. capsici zoospores in this study. The IOCM was provided in the forms of either

139

granular fragments or a packaged monolith (Figure 4.S2). To prevent clogging, coarse
sand (> 500 Âľm) layers of 2-3 cm were added to both ends of the sand filter and the
bottom of the granular IOCM column, but was not added in the monolith IOCM column
(Figure 4.S3).
4.2.2. Pathogen Culture and Inoculum.
P. capsici isolate SP98 was obtained from the culture collection of M. K.
Hausbeck at Michigan State University. The isolate was originally obtained from
pumpkin, is anA2 mating type, and is highly virulent to squash and other vegetable crops
5

. The culturing and production of zoospores were conducted per a previously established

method 30. Briefly, the P. capsici culture was grown on unclarified V-8 agar (UCV8) for
7–8 days. Sterile distilled water was added to the culture and incubated at 4°C for 30–45
minutes, and then at ambient temperature for 30 minutes so as to prompt the release of
zoospores from sporangia. The prepared zoospore suspension was transferred into a 2-L
Erlenmeyer flask. To determine the concentration of the zoospore suspension, a 1-mL
aliquot was placed into a 1.7-mL microcentrifuge tube, and vortexed for 70 seconds to
induce zoospore encystment. Afterwards, a 10-ÂľL aliquot of the encysted zoospore
suspension was pipetted onto a clean hemacytometer for enumeration (Bright-Line,
Hausser Scientific, Horsham PA). The concentration of the zoospore suspension was
adjusted to 4 × 105 zoospores/mL. The prepared suspension of motile biflagellate
zoospores was equally split into 500-mL capped bottles, and hand-shaken vigorously for
90 seconds to induce the zoospore encystment. The encysted zoospore suspension was
used to infest the irrigation water in the following greenhouse experiments.

140

4.2.3. Greenhouse Experiments.
Two greenhouse experiments were conducted to evaluate the effectiveness of
filtration to limit P. capsici transmission. The two experiments A and B differed in
duration and the type of IOCM used, lasting for 11 and 19 days, respectively. The
granular IOCM was used in experiment A, and the monolith IOCM in experiment B.
Experiment A included 6 treatments, including: 1) non-inoculated control without filter
(−Control); 2) inoculated control without filter (+Control); 3) non-inoculated with sand
filter; 4) non-inoculated with the granular IOCM filter; 5) inoculated with the sand filter;
6) inoculated with the granular IOCM filter. The 3rd and 4th treatments were included to
evaluate if the squash growth would be affected by the presence of the filter media.
Experiment B had 5 treatments, including: 1) non-inoculated control without filter
(−Control); 2) inoculated control without filter (+Control); 3) inoculated with the sand
filter; 4) inoculated with the IOCM monolith filter; 5) inoculated with the fungicide
etridiazole (Terrazole 35 WP; OHP, Mainland, PA) applied at the labeled rate (250 mg/L)
into the irrigation water in the holding tank prior to the initial inoculation. No filter was
used in the fungicide treatment, and fungicide was not applied to the other treatments.
Twelve-day-old seedlings of acorn squash ‘Table Ace’ (Cucurbita pepo) were
grown in 5-inch greenhouse pots with a peat potting mix (Suremix, SunGro, Galesburg,
MI) were used in the experiments. To ensure a sufficient number of replicates, 8–9 plants
were placed on each of the ebb-and-flow benches (Hummert, St. Louis, MO) in
experiment A, and 15 plants in experiment B. A 20-20-20 water-soluble fertilizer (Scotts
Company, OH) was added to the irrigation water, and the initial nutrient concentration
was 100 mg/L based on nitrogen. Groundwater was used as the source of irrigation water

141

and had a pH of 7.7 Âą 0.4 and electrical conductivity (EC) of 854 Âą 84 ÂľS. The pre-filter
tank in the filter treatments or the holding tank in the filter-free treatments was filled with
120 L of the fertilized irrigation water. Then, 500 mL of the prepared suspension of
encysted zoospores was added and agitated with a wooden dowel, resulting in a
concentration of 1.7 × 103 zoospores/mL. During the filtration, the irrigation water was
passed through the filter unit and then stored in the holding tank before the next
irrigation. The squash plants were irrigated for 10 minutes, twice each day to prevent
oversaturation of the growing media in the pots. To maintain a sufficient amount of water
during each experiment, the fertilized irrigation water was added when the water volume
decreased to about 70–80% of the initial volume. Water flow velocity and residence time
were calculated based on 3-day averages at the beginning of each experiment (n= 6).
During the experiments, the irrigation water was sampled from the holding tanks
periodically to determine pH, EC, nutrient levels, and zoospore concentrations. The pH
and EC of the irrigation water were measured to be 7.8 Âą 0.5 and 1.1 Âą 0.2 mS,
respectively, during the experimental periods. The water pressure inside the filter unit
was monitored in real time, along with the water temperature in the holding tank, and air
temperature and relative humidity in the greenhouse. The air temperature and relative
humidity were 28.9 ¹ 2.2 °C and 41.9 ¹ 8.6 %, respectively, in experiment A, and 25.7 ¹
3.1 °C and 16.6 ¹ 6.2 %, respectively, in experiment B (Figure 4.S4).
4.2.4. Filtration Performance Assessments
To evaluate the performance of the filtration systems in controlling P. capsici
outbreaks in squash, the plants were evaluated for foliar and root biomass at the end of
each experiment. At harvest, the roots were carefully washed, and fresh biomass of the

142

squash shoots and roots were measured. The shoot and root samples were then oven-dried
at 60°C for 3 days to determine dry biomass determined. In experiment A, the root
morphology was imaged and examined, and the zoospore concentration in the holding
tank was measured. Briefly, a 50 ml aliquot of water was collected from each tank
immediately after agitation, and returned to the laboratory. The suspension was
immediately filtered through 2.5 Îźm pore-size quantitative filter paper (GE Healthcare,
Pittsburg, PA), and the filter paper plated onto BARP (50 mg L-1 benomyl, 100 mg L-1
ampicillin, 30 mg L-1 rifampicin, 200 mg L-1 pentachloronitrobenzene)-amended CV8
(i.e., 100 mL V8 juice filtered through 4 layers of cheeses cloth, 3 g CaCO3, 16 g agar L1

). After 2 days, the filter paper was removed and colonies were enumerated. In

experiment B, the percetange of wilted plants was determined throughout the study to
illustrate infection progress.
4.2.5. Chemical Analyses
To assess if the IOCM released dissolved iron or iron oxide particles during the
experiments, 120 mL water samples were collected from the holding tank at the begging,
middle, and end of experiments. The collected samples were divided into two
subsamples. One set of subsamples were filtered through the 0.45-Âľm membrane filter
using a vacuum pump. Then 10 mL of each non-filtered or filtered water sample was
added into a 50-mL digestion tube, followed by the addition of 4 N HCl. The digestion
tubes were heated at 90°C for 3 hours using a digestion block (Magnum Series; Martin
Machine, IL) to completely dissolve iron oxides. Afterwards, the tube was filled with DI
water to 25 mL. The concentration of iron in the digested water samples was measured by
atomic absorption spectroscopy (AAnalyst 400, PerkinElmer, Waltham, MA).

143

To measure the Îś-potentials of IOCM, 1 gram was gently ground using a pestle
and mortar. The ground IOCM was ultrasonicated in 10 mL DI water for 10 minutes and
settled for 30 minutes. Then 1 mL of the supernatant was taken for the Îś-potential
measurements using a Malvern ZetaSizer Nano ZS (Malvern, Westborough, MA).
4.2.6. Statistical Analysis
Statistical analyses on the experimental data were performed with the R software
using the “LSD” R package for parametric test. Comparison among treatments were
made by one-way analysis of variance (p ≤ 0.05), When a significant F value was
determined, means were separated by the LSD’s multiple comparison test.

4.3. RESULTS
4.3.1. Irrigation and Filtration Systems
The water flow velocities for the IOCM and sand filters were 23.7 Âą 3.9 and 5.5 Âą
1.62 cm/min in experiment A, and 22.0 Âą 2.3 and 6.7 Âą 3.3 cm/min in experiment B.
Consequently, the water residence time for the IOCM and sand filters were 2.2 Âą 0.4 and
10.2 Âą 3.6 minutes in experiment A, and 2.2 Âą 2.32 and 9.4 Âą 4.5 minutes in experiment
B. Operating water pressure of the IOCM and sand filters was maintained at 10.5 Âą 6.6
kPa (i.e., 1.5 Âą 0.96 psi) and 9.1 Âą 1.9 kPa (i.e., 1.3 Âą 0.28 psi), respectively, under
typical greenhouse water temperatures (Figure 4.S5 and 4.S6). Because rapid sand
filtration often has a water velocity of 8.3–25 cm/min

35-37

, the IOCM filter allows a fast

flow rate. The water velocity in the sand filter was at least 13 times of that in typical slow
sand filtration (0.17–0.5 cm/min) 38. For this research, a high water flow was established
under low water pressure provides irrigation efficiency that can be advantageous to

144

greenhouses that need to deliver high water volume in a short time while conserving
energy use.
In terms of irrigation water quality, the concentration of NO3−, P, and Mg were
lower, and the concentration of K and Na were higher in the IOCM filter effluent,
compared with other treatments in experiment B (Table 4.S1). The isoelectric point of
iron oxide typically ranges from 7.5 to 9.0

39, 40

and the Îś-potential is 13.1 Âą 1 mV at pH

7.6. Therefore, the IOCM surface might be protonated and become positively charged at
the experimental pH of 7.8 Âą 0.5, thus providing sorption sites for the negatively charged
NO3− and phosphate ions. Additionally, the negatively charged surface sites in the
aluminosilicate material in the IOCM could also bind cation such as Mg2+, thus
decreasing its concentration. The increased K and Na concentrations likely resulted from
the release of these ions from the IOCM (Table 4.S1). Moreover, iron oxide particles
could be released from the granular IOCM in experiment A. The iron concentrations in
the non-filtered and filtered water samples were as high as 7 and 1.96 mg/L, respectively
(Table 4.S2). In contrast, the iron concentrations in the non-filtered and filter water
samples were 2.39 mg/L, at the maximum, and non-detectable in the monolith IOCM,
respectively (Table 4.S3). While the IOCM appeared to release iron, the iron levels in the
irrigation water would not cause plant phytotoxicity 41, 42. Also, this release may decrease
with time as less strongly attached iron nano particles will be washed off with repeat
washing.
The concentration of P. capsici in the irrigation water determined by colony
forming unit (CFU). The zoospore concentration in the irrigation water for the inoculated
sand and IOCM filter treatments was decreased by more than 90% from the concentration

145

in the inoculated control treatment (Table 4.S4), consistent previous studies

30

. The

zoospore concentration remained low for the duration of the experiment. At the final
zoospore measurement, the irrigation water in the sand filter treatment contained 83%
fewer zoospores than the inoculated control treatment, whereas the irrigation water in the
IOCM filter treatment had no zoospores.
4.3.2. Plant Assessment
In the inoculated control treatment, significant plant damage was observed at the
end of experiment A (Figure 4.1), and the squash plants were dead and dried at the end of
experiment B (Figure 4.2), due to a longer exposure to P. capsici. A visual change was
not observed among the squash plants in the non-inoculated sand filter and IOCM filter
treatments, compared with those in the non-inoculated control treatment. Thus, the filter
media did not negatively affect plant growth. In both experiments, 44–100% of the plants
in the sand filter treatment appeared to be infected by P. capsici, and exhibited wilting
and stunting (Figure 4.1 and 4.2). The plants in the IOCM filter treatments did not display
wilt symptom. The plants in the treatment that included etridiazole application to the
irrigation water in experiment B showed phytotoxicity symptoms including interveinal
chlorosis (Figure 4.S7). The visual appearance of the squash shoots were in accordance
with their root morphology (Figure 4.S8). In experiment A, the roots of all the plants in
the inoculated control treatment and 44% of the plant roots in the sand filter treatment
displayed root rots, whereas the plant roots in other treatments were healthy. The wilt
symptom of the plants was monitored throughout experiment B (Table 4.1). In the
inoculated control treatment, all of plants wilted and died 6 days after inoculation.
Disease development was slower in the sand filter treatment, and irreversible wilting was

146

observed 19 d after inoculation. No wilt symptoms were observed for plants in the other
treatments. Sand filtration slowed but did not completely prevent disease development in
the squash plants. The IOCM filter and fungicide treatments effectively prevented disease
symptoms.
The visual observations of the plants were in agreement with the plant biomass
measurements. In the absence of P. capsici, the weights of fresh and dry leaves and roots
in the sand and IOCM filter treatments were not significantly different from that in the
non-inoculated control treatment, confirming that the two filter media did not negatively
affect the squash growth. In experiment A, the fresh and dry weights of squash leaves and
roots in the sand and IOCM filter treatments were similar to those of plants in the noninoculated control treatment. The plants in the inoculated sand filter treatment had a
significantly lower foliar fresh weight (Figure 4.3). While the plants in the inoculated
sand filter treatment visually appeared inferior to those in the inoculated IOCM filter
treatment, the measured biomass was not significantly different. In experiment B, the
plant biomass in the inoculated IOCM filter treatment was significantly higher (p ≤ 0.05)
than that of the plants in the inoculated control and sand filter treatments. The foliar fresh
weight of the plants in the inoculated sand filter treatment was, on average, 72% less than
that of the inoculated IOCM filter treatment. There was no significant difference in the
plant biomass between the fungicide treatment and the sand and IOCM filter treatments.
The non-inoculated control treatment had significantly higher plant biomass than all other
treatments (Figure 4.4).

147

Figure 4.1. Images of squash plants at the end of the Experiment A (11 days after inoculation).

148

Figure 4.2. Representative images of squash plants at the end of the Experiment B (19 days after inoculation).

149

Table 4.1. Percentage of the plants with wilting symptoms in the Experiment B.
Days after inoculation
Treatment
4
5
6
10
11
12
13
14

15

16

17

19

‒Control

0

0

0

0

0

0

0

0

0

0

0

0

+Control

0

13

100

100

100

100

100

100

100

100

100

100

Sand filter

0

0

0

13

33

40

40

67

80

93

93

100

IOCM filter

0

0

0

0

0

0

0

0

0

0

0

0

Terrazole 35 WP

0

0

0

0

0

0

0

0

0

0

0

0

150

Figure 4.3. Squash foliar fresh (A) and dry (B) weight, and root fresh (C) and dry (D) weight in the Experiment A (LSD test, P
< 0.05).

151

Figure 4.4. Squash foliar fresh (A) and dry (B) weight in the Experiment B (LSD test, P < 0.05).

152

4.4. DISCUSSION
4.4.1. Filtration Performance
This results showed that the squash plants irrigated with water after fast-flow
IOCM filtration were of higher quality and biomass compared to plants in the sand
filtration treatment. This likely resulted from the increased removal of P. capsici
zoospores in the IOCM filter than in the sand filter. The improved disease control
observed in the IOCM filter treatment could have resulted from greater electrostatic
attraction of the negatively charged zoospores to the positively charged iron oxide surface
sites in the IOCM, compared to the negatively charged sand surface

30, 40, 43, 44

.The

biomass decrease in squash in the IOCM treatment probably resulted from nutrient
removal by the IOCM (Table 4.S1). The squash plants in the IOCM treatment displayed
chlorosis of older leaves and slight stunting symptoms. These are common symptoms of
nitrogen, phosphorus, and magnesium deficiency

45

. Thus, application of supplemental

nutrient in irrigation water or slow release fertilizer in pots would be needed when the
IOCM filter is used in filtering recycled nutrient solutions.
Sand filter media effectively limited P. capsici zoospores, but did not completely
prevent disease. Os et.al (1998) reported that physical factors such as pore straining is an
important mechanism in the removal of pythiaceous zoospores rather than surface charge.
Many previous studies conducted in laboratories and greenhouses have consistently
shown that sand filters can effectively remove pythiaceous zoospores from water
47

26-30, 46,

. However, in this study 100 % of the squash plants died 19 days after the initial

inoculation. It appears that despite the substantial removal of P. capsici zoospores, a
small percentage of the zoospores may have passed through the sand filter due to the

153

initial high pathogen inoculum concentration (1.7 × 104 zoospores/mL) 30. Indeed, it was
reported that Pythium sp. and Phytophthora sp. were efficiently removed when the water
flow was less than 0.5 cm/min and the pathogen loading was not high

38

. Jeon et. al.

(2017) confirmed that Pythium zoospores were effectively removed in the fast sand filter
at a low pathogen inoculum concentration of 68 zoospores/mL. The isolate of P. capsici
that was used is highly virulent and the squash used in the trial is highly susceptible

48

,

thus even a low concentration of transported zoospores could cause serious crop damage
29, 49

. Also, there was not time in this study for the rapid sand filters to develop a biofilm

(or Schmutzdecke) as each was conditioned with irrigation water one or two days before
inoculation. The active biofilm layer on the sand surface was considered the most
important factor for a high pathogen removal efficiency 50. Lee and Oki 26 reported that it
took more ten days to reach a 100% reduction of P. capsici using the slow sand filter.
However, others suggested that physicochemical factors (e.g., grain size, grain surface
properties, and solution chemistry) are more important in controlling the removal of
phythiaceous zoospores 28-31.
4.4.2. Fungicide Application
The fungicide treatment provided a comparison among the filtration treatments in
experiment B. The plant biomass from the fungicide treatment was not different from that
of the sand and IOCM treatments, but was significantly less than the non-inoculated
control. While wilting was not observed, the plant growth in the fungicide treatment was
negatively impacted, possibly due to fungicide phytotoxicity. Significant chlorosis was
observed on the foliage (Figure 4.S7). Other studies have indicated that etridiazole can
cause phytotoxicity symptoms in avocado seedlings, Douglas-fir seedlings, and black

154

pepper leaves

51-53

. Etridiazole is not registered for food crops but is commonly used in

the greenhouse, especially for control of Pythium spp. The fungicides mefenoxam (active
enantiomer of metalaxyl; Subdue Maxx; Syngenta Crop Protection, Greensborough, NC)
and etridiazole have historically been used to manage root rot of ornamentals in
greenhouse production
56-58

54-56

and provide effective control when applied as soil-drenches

. Resistance to mefenoxam has developed in greenhouse populations of Pythium and

Phytophthora due to repeated fungicide use 59, 60 55, 61. Greenhouses that recycle irrigation
water face additional challenges because sub-lethal levels of recycled fungicides can
exert selection pressure on pathogen populations to develop fungicide resistance

61, 62

.

Overall, in addition to the observed phytotoxicity, the fungicide application management
option is not ideal because: the availability of effective fungicides in the market is
limited; Phytophthora spp. often quickly develop fungicide resistance; and the fungicide
discharge in wastewater raises environmental concerns

16, 20

. Finally, the etridiazole

treatment may not be as effective against P. capsici as recently registered systemic
fungicides

63

. However, many of these fungicides are not labeled for greenhouse

vegetable production. Thus, it is critical to understand the target pathogen, the pathogen
loading, fungicide efficacy, and plant susceptibility in order to design an effective sand
filtration system and integrated disease management program.

4.5. IMPLICATIONS
Contamination of recirculating irrigation water by oomycete pathogens is a major
threat to greenhouse producers of vegetables and floriculture crops. Many greenhouse
operations recycle irrigation water due to environmental regulation and public concern

155

regarding water and pesticide use. Control of oomycete pathogens in recirculating
irrigation water is critical to the greenhouse industry. This study indicates that physical
removal of pathogens using fast-flow may be a promising tool for disease management in
the greenhouses. Previous studies using the fast-flow filtration to remove plant pathogens
from irrigation water have been limited. This study found that the IOCM filter could
effectively protect squash plants from P. capsici, but resulted in nutrient deficiency. The
sand filter did not prevent, but slowed the disease in the squash. Thus, it is clear that the
IOCM filter has the potential to be used in treating irrigation water in greenhouse
vegetable production, but nutrients will need to be supplied separately to the recirculating
irrigation water to avoid nutrient deficiency. Fast-flow filtration has the potential for
greenhouse disease management with future work needed to develop improved filtration
systems (i.e., filter media type, operation parameters, and process design) that limit plant
pathogens for an extended period while maintaining sufficient nutrient levels for plant
growth.

4.6. ACKNOWLEDGMENTS
This research was partly supported by American Floral Endowment, and
Michigan State University AgBioResearch, and a Subaward from MetaMateria
Technologies LLC through the Small Business Innovation Research Program
Competitive Grant No. 2015-33610-23704 from the USDA National Institute of Food
and Agriculture. The views and opinions of the authors expressed herein do not
necessarily reflect those of funding organizations and Michigan State University.
Mention of tradenames or commercial products does not constitute endorsement or

156

recommendation for use.

157

APPENDIX

158

APPENDIX
SUPPORTING INFORMATION

Figure 4.S1. Scheme of the ebb-and-flow irrigation system constructed in the greenhouse.

159

Figure 4.S2. Granular (A) and monolith (B) iron oxide coated media (IOCM).

160

Figure 4.S3. Filter design of granular IOCM (A), sand (B), and monolith IOCM (C).

161

Figure 4.S4. Air temperature and relative humidity during the Experiment A (A) and Experiment B (B) in the greenhouse.

162

Figure 4.S5. Water pressure in the sand filter in the Experiment A (A) and in the sand and IOCM filters in the Experiment B
(B).

163

Figure 4.S6. Water temperature in the holding tank during the Experiment A (A) and Experiment B (B).

164

Table 4.S1. Nutrient concentrations in irrigation water during the Experiment B.
NO3-

P

K

Ca

Mg

Na

Treatments
mg/L
Days a
3
10 19
3
10 19
3
10 19
3
10 19
3
10 19
- Control
29 30 51 24 17.6 21.1 93 89 110 41 55 68 38 35 41
+ Control
16 32 56 12.8 16 17.6 53 84 120 27 41 55 30 29 36
Sand filter
27 24 35 19 13.2 11 84 72 80 55 55 68 33 30 36
IOCPM filter
17 1.9 0.4 0.1 2.1 0.1 123 111 137 68 68 55 17 19 19
Etridiazole
28 24 27 12 12.8 6.6 89 78 87 55 41 41 30 29 35
Ground water
0 ND ND 0.1 ND ND 2 ND ND 123 ND ND 35 ND ND
a
The numbers in the “days” means the days from the start of the experiment (i.e., the inoculation).
b
ND means “no data available”

165

3
18
11
18
54
24
18

10
20
16
16
46
19
ND

19
22
23
19
52
23
ND

Table 4.S2. Iron concentrations in the filtered and non-filtered irrigation water in the Experiment A.
5 days (August 1) a
11 days (August 7)
Filtered
Non-filtered
Filtered
Non-filtered
mg/L
b
−Control
NA
0.19
ND c
0.50
Sand filter no P.
NA
0.31
0.16
0.02
IOCM filter no P.
NA
0.63
0.21
0.47
No filter with P.
NA
0.44
0.04
0.34
Sand filter with P.
NA
0.32
LOD
0.08
IOCPM filter with P.
NA
7.00
1.96
6.43
a
Days means the days after the inoculation, and the sampling date in 2014 is provided in the parentheses.
b
NA means “no data available” due to missing of filtration procedure.
c
ND means the concentrations below the limit of detection.

166

Table 4.S3. Iron concentrations in the filtered and non-filtered irrigation water in the Experiment B
Exp. A
3 days (March 4) a
10 days (March 11)
19 days (March 20)
Filtered
Non-filtered
Filtered
Non-filtered
Filtered
Non-filtered
- Control
+ Control
Sand filter
IOCPM filter
Terrazole
a
b

ND b
ND
ND
ND
ND

ND
ND
0.02
0.61
ND

ND
ND
ND
ND
ND

ND
ND
ND
2.39
ND

ND
ND
ND
ND
ND

Days means the days after the inoculation, and the sampling date in 2015 is provided in the parentheses.
ND means the concentration below the limit of detection.

167

ND
ND
ND
0.42
ND

Table 4.S4. Zoospore concentrations in the Experiment A by colony forming units a.
Days (Sampling Date) b
Treatments
0 (July 27)
2 (July 29)
- Control
Sand filter no P.
IOCM filter no P.
No filter with P.
Sand filter with P.
IOCPM filter with P.
a
b

0
0
0
6.7
6.1
4.1

6 (August 2)

0
0
0
20.4
0.2
0.4

Colony forming units (CFU) = CFU/mL
Days means the days after the inoculation, and the sampling date in 2014 is provided in the parentheses.

168

0
0
0
2.4
0
0.4

Figure 4.S7. Leaf and stem images of the squash plants in the Terrazole 35WP treatment
(A and B) and the non-inoculated control treatment (‒Control, C and D).

169

Figure 4.S8. Root images at the end of the experiment A (i.e., 11 days after inoculation).

170

S1. Squash Greenhouse Experiments Raw Data
Table 4.S5. Experiment A: Squash foliar fresh weight (g)
Number

- Control

Sand filter
no P.

IOCM
filter no P.

No filter
with P.

Sand filter
with P.

1
2
3
4
5
6
7
8
9
Mean
SD

32.8
34.7
33.7
38.6
27.4
35.2
34.7
22.6
32.9
32.5
4.5

34.3
39.1
35.3
34.9
31.3
36.0
35.8
39.3
N.D
35.7
2.4

36.2
38.1
40.3
30.2
36.8
39.5
38.2
N.D
N.D
37.0
3.1

6.9
5.0
15.1
8.7
9.1
7.0
10.9
9.7
N.D
9.1
2.9

10.1
23.7
33.4
20.4
10.8
22.5
19.1
31.8
29.4
22.4
7.9

IOCM
filter with
P.
20.4
33.4
34.5
31.4
28.6
35.3
36.8
27.6
30.7
31.0
4.7

Table 4.S6. Experiment A: Squash foliar dry weight (g)
Number

- Control

Sand filter
no P.

IOCM
filter no P.

No filter
with P.

Sand filter
with P.

1
2
3
4
5
6
7
8
9
Mean
SD

2.5
2.5
2.5
2.8
2.0
2.6
2.5
2.6
2.5
2.5
0.2

2.7
2.9
2.7
2.6
2.2
2.7
2.7
2.9
N.D
2.7
0.2

2.8
2.9
3.1
2.3
2.9
3.1
2.9
N.D
N.D
2.8
0.3

1.0
0.9
1.5
1.1
1.1
0.8
1.4
0.9
N.D
1.1
0.2

1.3
2.1
2.7
2.1
1.3
2.1
1.7
2.5
2.5
2.0
0.5

continue

171

IOCM
filter with
P.
1.8
2.6
2.9
2.5
2.2
2.7
2.8
2.2
2.4
2.4
0.3

Table 4.S7. Experiment A: Squash root fresh weight (g)
Number

- Control

Sand filter
no P.

IOCM
filter no P.

No filter
with P.

Sand filter
with P.

1
2
3
4
5
6
7
8
9
Mean
SD

3.9
4.8
3.8
4.2
3.0
4.9
4.3
3.0
4.3
4.0
0.6

4.8
4.2
3.7
4.2
3.3
3.9
4.3
4.4
N.D
4.1
0.4

5.4
5.6
6.2
4.5
4.6
5.8
4.9
N.D
N.D
5.3
0.6

0.5
0.4
1.0
0.7
0.8
0.8
0.8
1.0
N.D
0.7
0.2

1.1
3.3
4.2
4.2
2.1
1.0
2.4
2.8
3.7
2.8
1.1

IOCM
filter with
P.
3.5
4.5
5.4
5.7
3.6
4.6
5.4
4.4
4.8
4.7
0.7

Table 4.S8. Experiment A: Squash root dry weight (g)

Number

- Control

Sand filter
no P.

IOCM
filter no P.

No filter
with P.

Sand filter
with P.

1
2
3
4
5
6
7
8
9
Mean
SD

0.33
0.32
0.29
0.33
0.24
0.36
0.36
0.22
0.34
0.31
0.05

0.32
0.33
0.27
0.30
0.26
0.28
0.31
0.34
N.D
0.30
0.03

0.39
0.40
0.44
0.31
0.37
0.42
0.39
N.D
N.D
0.39
0.04

0.11
0.08
0.18
0.13
0.13
0.12
0.15
0.13
N.D
0.13
0.03

0.20
0.34
0.32
0.35
0.32
0.16
0.32
0.30
0.34
0.29
0.06

continue

172

IOCM
filter with
P.
0.26
0.34
0.37
0.40
0.28
0.32
0.41
0.31
0.35
0.34
0.05

Table 4.S9. Experiment B: Squash foliar fresh weight (g)
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

- Control
108.1
111.0
106.8
96.4
91.9
114.9
111.8
92.7
103.1
116.5
98.4
113.2
113.5
109.3
111.1
106.6
7.8

+ Control
2.3
1.2
1.1
1.9
1.5
1.4
1.5
1.2
1.7
1.5
2.2
1.5
1.4
1.9
2.1
1.6
0.4

Sand filter
21.2
9.8
3.5
6.2
28.0
14.8
12.8
23.1
21.6
47.3
8.1
9.3
12.9
10.9
10.3
16.0
10.7

IOCM filter
59.3
50.5
70.7
61.7
56.1
31.6
61.5
67.5
62.0
50.6
52.1
54.7
64.3
57.6
51.3
56.8
9.0

Etridiazole
48.1
46.0
41.6
43.0
39.5
38.1
41.0
41.0
41.9
41.5
41.7
52.1
41.1
39.9
33.0
42.0
4.2

IOCM filter
6.2
5.1
7.2
6.1
5.9
5.1
6.5
7.0
6.7
5.2
5.5
5.9
7.0
6.1
5.7
6.1
0.7

Etridiazole
4.8
4.4
4.2
4.2
4.1
4.2
4.2
4.1
4.5
4.1
4.1
5.1
4.2
3.9
3.6
4.2
0.3

Table 4.S10. Experiment B: Squash foliar dry weight (g)
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Mean
SD

- Control
9.6
9.7
9.8
8.4
8.2
10.9
9.7
8.0
9.7
10.4
8.8
10.6
10.2
9.5
10.6
9.6
0.9

+ Control
1.4
1.0
0.9
1.2
1.1
1.2
1.1
1.1
1.2
1.2
1.3
1.0
0.9
1.3
1.3
1.1
0.1

Sand filter
4.2
2.9
2.0
2.3
4.9
3.6
3.3
4.1
4.4
6.4
3.0
3.6
4.1
3.6
3.4
3.7
1.0

continue

173

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174

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CHAPTER 5

CONCLUSION AND FUTURE WORK
In this work, effective methods for the vegetable and floriculture industries to
treat irrigation water and remove and/or reduce water mold pathogens (Phytophthora and
Pythium zoospores) were investigated, as well as the pathogen retention mechanisms in
porous media. A laboratory column study was performed to investigate the effect of filter
media properties, solution chemistry, and zoospore encystment on the zoospore transport.
Greenhouse experiments were then conducted to demonstrate the proof-of-concept of
using fast-flow filtration systems to control the pathogen infections in susceptible host
plants (i.e., poinsettia and squash). Major conclusions were outlined as follows.
I. The transport of Phytophthora capsici zoospores in porous media was
collectively controlled by surface properties of zoospores and porous media, and solution
chemistry. Significantly more encysted zoospores were retained in IOCS than in uncoated
sand, and at pH 4.4 than at pH 7.2, which likely resulted from increased electrostatic
attraction between zoospores and grain surfaces. At pH 7.2, up to 99% and 96% of the
encysted zoospores were removed in IOCS and uncoated sand, respectively, due to a
combination of strong surface attachment, pore straining, and adhesive interactions.
Motile biflagellate zoospores were more readily transported than encysted zoospores,
thus posing a greater dispersal and infection risk.
II. The fast-flow sand filtration with low water pressure consistently removed
Pythium aphanidermatum zoospores, and no differences in root rot severity, height,
biomass, and horticultural rating were observed for the plants in the sand filter treatment,
compared with the non-inoculated control plants. However, the activated carbon filter
181

removed essential nutrients from the irrigation water, resulting in plant nutrient
deficiency and consequently leaf chlorosis and lower plant biomass, height, and
horticultural ratings. Overall, the rapid sand filter has the potential to be used for
controlling Pythium root rot in greenhouses, but maintenance was required to prevent
clogging.
III. The IOCM filter could effectively protect squash plants from Phytophthora
capsici, but caused the nutrient deficiency in the squash. The sand filter could not prevent,
but only slow the disease development in the squash. The IOCM filter may be used to
control Phytophthora infections in greenhouse vegetable production if the nutrients can
be supplied separately instead of through irrigation water.
Overall, the results suggest that physical removal of pathogens using fast-flow
filtration can overcome many limitations of fungicide application, and may be a
promising alternative for disease management in greenhouses. Building upon this work,
future research will need to focus on assessing the longevity of the system performance
by optimizing filter media and operation parameters. The filter design can be further
improved to reduce clogging of the sand filter. Operation parameters such as irrigation
frequency and duration can be optimized to provide adequate water to crops while
minimizing the pathogen infection and improving the longevity of the filtration systems.
Finally, future work should be conducted in large-scale systems to assess the
performance, optimize operation parameters, and analyze operation costs in commercial
greenhouses.

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