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Effects of temperature and hydrogen ion concentration on disease caused by Fusarium solani f. sp. phaseoli in Vigna radiata grown in hydroponic nutrient solution

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Title:
Effects of temperature and hydrogen ion concentration on disease caused by Fusarium solani f. sp. phaseoli in Vigna radiata grown in hydroponic nutrient solution
Creator:
Schuerger, Andrew Conrad, 1956-
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English
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xiii, 243 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Agglutination ( jstor )
Diseases ( jstor )
Fusarium ( jstor )
Hydroponics ( jstor )
Inoculation ( jstor )
Inoculum ( jstor )
Nutrient solutions ( jstor )
pH ( jstor )
Plant roots ( jstor )
Sugars ( jstor )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1991.
Bibliography:
Includes bibliographical references (leaves 221-242).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Andrew Conrad Schuerger.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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AJF5160 ( NOTIS )
26181208 ( OCLC )

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EFFECTS OF TEMPERATURE AND HYDROGEN ION CONCENTRATION ON
DISEASE CAUSED BY FUSARIUM SOLANI F. SP. PHASEOLI IN VIGNA
RADIATA GROWN IN HYDROPONIC NUTRIENT SOLUTION












by


ANDREW CONRAD SCHUERGER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1991













ACKNOWLEDGMENTS


The author would like to express his sincere gratitude

to Dr. David J. Mitchell for his constant patience, honesty,

and moral support during the course of this project. This

research certainly would have been more difficult if it were

not for the friendship that Dr. Mitchell extended to that

young student with the unusual ideas so many years ago. The

author also would like to thank the other members of the

supervisory committee, Drs. R. Charudattan, James O.

Strandberg, and David M. Sylvia, for their patience and

suggestions. Special thanks are due Dr. David T. Kaplan for

his suggestions in the experiments on spore attachment to

plant roots.

Appreciation is extended to the Walt Disney World, Co.,

which has provided financial support for this research, and

to the Biological Science Office of the National Aeronautics

and Space Administration (NASA) at the Kennedy Space Center,

FL, which has provided in-kind support under a joint

agreement between the Walt Disney World, Co., and NASA. In

addition, thanks are due Dr. Henry A. Robitaille, Manager of

Science and Technology, EPCOT Center, and Dr. William M.

Knott, III, Biological Science Officer, NASA, Kennedy Space

Center, for their suggestions and support for this research.

Special thanks are given to Dr. Frederick L. Petitt,

Manager of The Land, EPCOT Center, and Steven Linda,










Department of Statistics, University of Florida, for their

suggestions and assistance in the analysis of experimental

data. Their assistance greatly improved the quality of this

research. Furthermore, appreciation is extended to Dinah

Jordan, for her assistance in the preparation of this

manuscript, and to the plant pathology staff at The Land,

Jean Carlson-Batzer, William Hammer, Kristin Pategas, and

Bret Norman, who performed their jobs with the excellence

that created the freedom to pursue this project. Thanks

also are due the plant pathology students, Karen Wendt,

Andrew Deckard, Bryan Kleiwer, Phil Liable, Andrew Hoetger,

and James Adams, who helped in various ways.

The author also would like to acknowledge the

contributions of scientists and philosophers, both living

and deceased, who have provided the intellectual and

metaphysical framework of human knowledge. We seldom

recognize that our progress would be nearly futile if it

were not for the passions and tribulations of those who have

come before us.


iii















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS..................................... ii

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

LIST OF FIGURES........................................ ix

ABSTRACT ...... .................. .. ............... xi

CHAPTERS

1 INTRODUCTION........ ............................. 1

2 EFFECTS OF CULTURE MEDIA ON MACROCONIDIUM
MORPHOLOGY AND PATHOGENICITY OF FUSARIUM SOLANI
F. SP. PHASEOLI.............. .......... .......... 11

Introduction............... ...... ............ 11
Materials and Methods............................ 12
Results.... ................................. ...30
Discussion....................... ........... ..... 38

3 EFFECTS OF TEMPERATURE, HYDROGEN ION
CONCENTRATION, HUMIDITY, AND LIGHT QUALITY ON
DISEASE CAUSED BY FUSARIUM SOLANI F. SP.
PHASEOLI IN VIGNA RADIATA ....................... 42

Introduction................... ................. 42
Materials and Methods............................. 45
Results......... .......... ........ 54
Discussion.......................... .. ........... 87

4 EFFECTS OF TEMPERATURE AND HYDROGEN ION
CONCENTRATION ON ATTACHMENT OF MACROCONIDIA OF
FUSARIUM SOLANI F. SP. PHASEOLI TO ROOTS OF VIGNA
RADIATA IN HYDROPONIC NUTRIENT SOLUTION ......... 102

Introduction..................................... 102
Materials and Methods............................ 104
Results.............. ........ ............... 115
Discussion.............. ......... ............ 128










5 THE INFLUENCE OF SECRETED SPORE MUCILAGE OF
MACROCONIDIA OF FUSARIUM SOLANI F. SP.PHASEOLI
ON SPORE ATTACHMENT TO ROOTS OF VIGNA
RADIATA IN HYDROPONIC NUTRIENT SOLUTION.......... 141

Introduction................ ............ ..... 141
Materials and Methods........................... 143
Results............................ ... 148
Discussion...................................... 166

6 EFFECTS OF CARBON SOURCE ON SPORE ATTACHMENT AND
GERMINATION OF MACROCONIDIA OF FUSARIUM SOLANI
F. SP. PHASEOLI ON ROOTS OF VIGA RADIATA IN
HYDROPONIC NUTRIENT SOLUTION.................... 173

Introduction.................................. 173
Materials and Methods........................... 176
Results.................. .............. .. 184
Discussion.............................. ......... 193

7 SUMMARY AND CONCLUSIONS......................... 199

APPENDICES

A DRY WEIGHTS AND SHOOT-ROOT RATIOS FROM
PATHOGENICITY TESTS........................... 208

B DRY WEIGHTS AND SHOOT-ROOT RATIOS FROM
SPORE ATTACHMENT EXPERIMENTS.................. 217

LITERATURE CITED................................... 221

BIOGRAPHICAL SKETCH................................... 243














LIST OF TABLES


Table Page

2-1. Production of macroconidia, microconidia, and
chlamydospores by 43 strains of Fusarium spp.
grown for 14 days on modified carnation leaf
agar (MCLA), carnation leaf agar (CLA), and
potato dextrose agar (PDA)...................... 15

2-2. Fusarium spp. tested for pathogenicity on
various hosts in a hydroponic system............. 20

2-3. Length and width of macroconidia of Fusarium
solani f. sp. phaseoli grown on four
different media................................. 36

2-4. Effects of culture media on the virulence of
inoculum of Fusarium solani f. sp. phaseoli and
on disease in Vigna radiata..................... 37

3-1. Effects of temperature on disease caused by
Fusarium solani f. sp. Dhaseoli in
Vigna radiata.................................. 56

3-2. Effects of hydrogen ion concentration and
temperature on disease caused by Fusarium
solani f. sp. phaseoli in Vigna radiata......... 57

3-3. Effects of humidity and temperature on disease
caused by Fusarium solani f. sp. phaseoli in
Viqna radiata......................... ......... 58

3-4. Effects of light quality and temperature on
disease caused by Fusarium solani f. sp.
phaseoli in Vigna radiata...................... 59

3-5. Effects of temperature on the colonization
of roots of Vigna radiata by Fusarium solani
f. sp. phaseoli................................. 67

3-6. Effects of hydrogen ion concentration and
temperature on the colonization of roots of
Vigna radiata by Fusarium solani f. sp. phaseoli 68

3-7. Effects of humidity and temperature on the
colonization of roots of Vigna radiata by
Fusarium solani f. sp. phaseoli................. 69









3-8. Effects of light quality and temperature on
the colonization of roots of Vigna radiata by
Fusarium solani f. sp. phaseoli................. 70

3-9. Effects of temperature on the numbers of propa-
gules of Fusarium solani f. sp. phaseoli detected
in nutrient solution over time during experiments
on disease development in Vigna radiata......... 72

3-10. Effects of hydrogen ion concentration and
temperature on the numbers of propagules of
Fusarium solani f. sp. phaseoli detected in
nutrient solution over time during experiments on
disease development in Vigna radiata............. 73

3-11. Effects of humidity and temperature on the numbers
of propagules of Fusarium solani f. sp. phaseoli
detected in nutrient solution over time during
experiments on disease development in
Viqna radiata................................... 74

3-12. Effects of light quality and temperature on the
numbers of propagules of Fusarium solani f. sp.
phaseoli detected in nutrient solution over time
during experiments on disease development in
Vigna radiata................................. .. 75

3-13. Effects of temperature on the elemental
composition of foliage of Vigna radiata in
experiments on disease development by Fusarium
solani f. sp. phaseoli......................... 78

3-14. Effects of hydrogen ion concentration and
temperature on elemental composition of foliage
of Vigna radiata in experiments on disease
development by Fusarium solani f. sp. phaseoli.. 80

3-15. Effects of humidity and temperature on the
elemental composition of foliage of Vigna radiata
in experiments on disease development by
Fusarium solani f. sp. phaseoli................. 82

3-16. Effects of light quality and temperature on the
elemental composition of foliage of Vigna
radiata in experiments on disease development
by Fusarium solani f. sp. phaseoli.............. 84

4-1. Attachment of macroconidia of Fusarium solani
f. sp. phaseoli to four different root orders
and to the lower hypocotyl of Vigna radiata...... 118

4-2. Effects of temperature and hydrogen ion
concentration on the attachment of macroconidia


vii









of Fusarium solani f. sp. phaseoli to
second-order roots of Vigna radiata............ 120

4-3. Effects of temperature and hydrogen ion
concentration on germtube elongation of Fusarium
solani f. sp. phaseoli after spore attachment to
second-order roots of Viana radiata............. 122

4-4. Effects of temperature and inoculum density on
the percentages of wilted plants of Vicna
radiata infected by Fusarium solani
f. sp. phaseoli........... ..................... 125

4-5. Effects of hydrogen ion concentration at the
time of inoculation on disease caused by
Fusarium solani f. sp. phaseoli
in Viqna radiata................................ 127

4-6. Effects of hydrogen ion concentration at the
time of inoculation on the numbers of propagules
of Fusarium solani f. sp. phaseoli detected in
nutrient solution over time in experiments on
disease development in Vigna radiata............ 128

6-1. Effects of carbon source on the agglutination
of macroconidia of Fusarium solani f. sp.
phaseoli in nutrient solution and
root homogenate................................ 179

6-2. Effects of macroconidium age on the agglutin-
ation of spores of Fusarium solani f. sp.
phaseoli in nutrient solution after incubation
of macroconidia in root homogenate.............. 185

6-3. Effects of hapten sugars on spore agglutination
and attachment of macroconidia of Fusarium
solani f. sp. phaseoli to roots of
Vigna radiata.................................. 187


viii













LIST OF FIGURES


Figure Page

2-1. Comparison of macroconidia of Fusarium solani
f. sp. phaseoli mounted on nutrient solution
agar and in a lactic acid, water, and
glycerol solvent ............................... 24

2-2. Schematic drawing of a cross section through
a seed germination tray used for the
production of seedlings of Vigna radiata ....... 27

2-3. Comparison of macroconidia of Fusarium solani
f. sp. phaseoli produced on four different
culture media................................... 34

3-1. Conidiophores of Fusarium solani f. sp.
phaseoli observed on severely damaged roots
of Vigna radiata................................ 63

3-2. Deformed conidiophores of Fusarium solani
f. sp. phaseoli on roots of Viana radiata grown
at 20 C and pH 4.0 ............................. 65
-l -
3-3. Spectral photon fluxes (Amol-s-.m-2) for
high pressure sodium, metal halide, and
fluorescent lamps............................... 97

4-1. In vitro effects of temperature on radial
growth and germtube length of Fusarium solani
f. sp. phaseoli................. ................ 116

4-2. In vitro effects of hydrogen ion concentration
on radial growth and germtube length of Fusarium
solani f. sp. phaseoli.......................... 117

4-3. Effects of inoculum density of Fusarium solani
f. sp. phaseoli and temperature on the severity
of disease in Vigna radiata..................... 124

5-1. Effects of culture age on cytology of macro-
conidia of Fusarium solani f. sp. phaseoli....... 150

5-2. Effects of age on the fixation and preparation
of macroconidia of Fusarium solani f. sp.
phaseoli for scanning electron microscopy....... 153









5-3. Agglutination of macroconidia of Fusarium solani
f. sp. phaseoli incubated in nutrient solution
and root homogenate............................ 155

5-4. Attachment of macroconidia of Fusarium solani
f. sp. phaseoli to root surfaces of
Vigna radiata.................... ............. 158

5-5. Agglutination of macroconidia of Fusarium
solani f. sp. phaseoli in root homogenate
maintained at 25 C and at different hydrogen
ion concentrations.............................. 160

5-6. Attachment of refuse from cultures of modified
carnation leaf agar (MCLA) to macroconidia of
Fusarium solani f. sp. phaseoli when
macroconidia were incubated in root homogenate.. 163

5-7. Production of spore mucilage at the tips of
macroconidia of Fusarium solani f. sp. phaseoli
during germination of conidia in root homogenate 166

6-1. Macroconidia of Fusarium solani f. sp. phaseoli
observed in the solutions of hapten sugars and
root homogenate ............................... 190

6-2. Macroconidia of Fusarium solani f. sp. phaseoli
observed after 24 hr in 50-mM solutions of
hapten sugars.................................... 192













Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
requirements for the Degree of Doctor of Philosophy


EFFECTS OF TEMPERATURE AND HYDROGEN ION CONCENTRATION ON
DISEASE CAUSED BY FUSARIUM SOLANI F. SP. PHASEOLI IN VIGNA
RADIATA GROWN IN HYDROPONIC NUTRIENT SOLUTION

by

Andrew Conrad Schuerger

August, 1991

Chairman: Dr. David J. Mitchell
Major Department: Plant Pathology


Disease caused by Fusarium solani f. sp. phaseoli in

Viana radiata was greatest at 20 C and decreased with

increasing temperature until plant weight differences

between noninoculated and inoculated plants were not

significant at 30-36 C. The numbers of propagules of the

pathogen recovered from nutrient solution at 20 C increased

significantly between 10 and 14 days after inoculation, but

not at higher temperatures. Disease development was not

affected by humidity or light quality. Results of

bioassays, based on root lengths of germinating seedlings of

Lactuca sativa, did not support the hypothesis that plant

growth-inhibiting or growth-promoting compounds accumulated

in nutrient solution.

The effects of [H+] on disease development varied with

the timing of the [H+] treatments. In one experiment,

plants were inoculated at pH 6.0, and after 24 hr the

xi













nutrient soultions in separate treatments were adjusted to

pH 4.0, 5.0, 6.0, or 7.0. In a second experiment, plants

were inoculated at pH 4.0, 5.0, 6.0, or 7.0, and after 24 hr

the nutrient solutions in all treatments were adjusted to

pH 6.0. When the [H+] was adjusted to different levels

after inoculation, disease was less at pH 4.0 than at pH

5.0, 6.0, and 7.0. When the [H+] was adjusted to different

levels during inoculation, disease was less at pH 7.0 than

at pH 4.0, 5.0, and 6.0. The effects of (H+] on the

severity of disease in the second experiment coincided with

the effects of [H+] on spore attachment to roots of V.

radiata; both disease and the numbers of spores attached to

roots were greatest at pH 4.0.

Spore attachment to root surfaces, agglutination of

macroconidia in a root homogenate, secretion of a spore

mucilage during germination, and accretion of refuse to

spore tips were all suppressed at pH 3.0 and 7.0, and at

35 C. Based on the these responses, it was proposed that an

inducer may be present in roots of V. radiata that may be

involved in spore attachment, agglutination, and

germination. It was hypothesized that the inducer might be

a plant lectin. However, hapten sugars of plant lectins

failed to stimulate spore agglutination in nutrient

solution, agglutination in root homogenate, or spore

attachment to roots. Thus, the results did not support the


xii













hypothesis that lectins were involved in the attachment of

macroconidia of F. solani f. sp. phaseoli to roots of V.

radiata.


xiii















CHAPTER 1
INTRODUCTION


Controlled ecological life support systems (CELSS) have

been proposed for space bases to regenerate oxygen, water,

and food from human and industrial wastes (18,133,166).

Biological processes will be combined with physical and

chemical technologies to provide life support systems that

will reduce requirements for the resupply of materials from

Earth. Higher plants are considered the primary candidates

for photosynthesis in CELSS (85,101,166,178,219). Wheat,

rice, white potatoes, sweet potatoes, soybeans, peanuts,

lettuce, and sugar beets have been proposed as the primary

crops for CELSS (219). Hoff et al. (101) suggested several

additional species, including herb and fruit species, that

would increase the diversity of the crew's diet. Algal

species also may provide a portion of the crew's diet, but

no more than 10% of the human diet can be supplied by algae-

derived food products without physiological problems (166).

The National Aeronautics and Space Administration's

CELSS Breadboard Project at the Kennedy Space Center, FL,

was established to determine the best methods for growing

plants in a multispecies semi-closed system (178). A 7.5-m

x 3.5-m vessel, called the Biomass Production Chamber, was










modified for plant production, and research was initiated in

1986 with wheat. The production of edible biomass, the

regeneration of air and water, and the development of

monitoring and control systems were among the primary

objectives of the project (178).

Various methods of plant production in CELSS have been

proposed (30,56,166,178,235), but the precise method to be

used in a CELSS will depend on the specific mission. For

example, a plant production system in a microgravity

environment may require more stringent controls for the

containment of nutrient solution than a plant production

system might require on the moon. Wright et al. (235)

proposed a membrane system for growing plants in

microgravity that was based on the capillary forces observed

between fluids and solids. Plant roots were grown adjacent

to one side of a microporous acrylic membrane, and a

nutrient solution was maintained as a recirculating fluid on

the other side of the membrane. The laminar-flow of the

fluid along the inside surface of the membrane prevented

leakage of the fluid into the root environment by inducing a

slight negative pressure at the fluid-membrane interface.

When plant roots contacted the membrane, capillary forces

between the roots and the nutrient solution would draw the

fluid through the membrane, subsequently wetting the plant

roots. Dreschel and Sager (56) developed a similar system

using porous ceramic tubes. In addition, aeroponic methods,

nutrient film technique (NFT), and substrated hydroponic










systems have been proposed for plant production in

microgravity (30,178,166). Lunar regolith and synthetically

manufactured zeolites have been proposed as solid substrates

for plant production at a moon base (151). However, liquid

hydroponic systems appear to have several advantages over

solid substrates (30,166).

Advantages of liquid hydroponic systems compared to

substrated hydroponic systems may include 1) an increase in

nutrient and oxygen availability to roots in a liquid

system, 2) a reduction in root stress due to lower physical

resistance in a liquid matrix, 3) greater dilution of root

secretions in the nutrient solution, and 4) the ease of

recovery of the root biomass to facilitate rapid nutrient

recycling in a CELSS (30). The greatest disadvantage of

substrated hydroponic systems is the cost of lifting the

substrate into orbit (166). In addition, handling the

substrate during harvest and planting activities may be

difficult in microgravity (166). Thus, hydroponic systems

have been proposed as the primary plant production method in

CELSS (30,101,124,166,219).

Plants in CELSS may be threatened by microorganisms

transported into space as contaminants on spacecraft

components, equipment, astronauts, and plant-propagative

materials. Extensive microbial contamination has been

reported in American spacecraft (24,131,180,181,215,

217,218,228). Species of Alternaria, Aspergillus, Candida,

Cephalosporium, Cladosporium, Fusarium, Mucor, Penicillium,










Phoma, Saccharomyces, and Trichoderma were the most

prevalent fungi, and species of Bacillus, Escherichia,

Klebsiella, Micrococcus, Pseudomonas, Staphylococcus,

Serratia, and Streptococcus were the most prevalent bacteria

recovered from these systems. Strict quarantine procedures

were not effective in preventing contamination during these

missions. For example, 57 genera of fungi and actinomycetes

were recovered from human and spacecraft surfaces during the

Apollo 14 and 15 moon missions (218); contamination occurred

despite 3 weeks of quarantine for the crew and spacecraft

prior to the missions (215,216).

Whether microbial contamination of CELSS modules will

constitute a realistic threat to plant health is difficult

to ascertain. New concepts in microbial ecology and plant

pathology will undoubtedly be developed for CELSS.

Microgravity may alter aspects of plant-microbe interactions

such that new and unique pathological relationships develop.

Microgravity has been shown to influence the development,

cytology, and physiology of plants (46,87,156,155,121,202).

For example, the lignin content of developing hypocotyls and

the cell density of root tips were reduced when mung bean

seed were germinated in microgravity (46,121). In an

accompanying experiment, root-cap cells of mung beans failed

to develop normally in microgravity (202); tissues were

poorly organized and cells appeared collapsed and degraded.

Furthermore, Moore et al. (156) demonstrated that decapped

roots of Zea mays L. failed to regenerate their caps in










microgravity. Perturbed shoot and root orientation has been

observed repeatedly in plants grown in space (87). And

lastly, changes in the structure of mitochondria,

dictyosomes, nuclei, and plastids have been noted in pea and

wheat plants grown in microgravity (87).

The physiology and development of microorganisms also

may be altered by microgravity (2,139,170,214,240). For

example, circadian rhythms in Neurospora crassa Shear &

Dodge (214) and Actinomyces levoris Kras (2), and the

development of fruiting bodies of the basidiomycete,

Polyporus brumalis (Pers.) Fris. (240), were disrupted in

microgravity. An increase in cell density was observed in

liquid cultures of Salmonella typhimurium (Loeffler) Cast. &

Chal. exposed to microgravity, which indicated that the

space-flown cells grew faster than the ground controls

(139); a similar effect of microgravity on cell density was

not observed with Escherichia coli (Migula) Cast. & Chalm.

Although no studies on plant-microbe interactions in

microgravity have appeared in the literature, clinostats

(193) have been used to test the effects of gravity

compensation on plant disease development (48,89,119,232).

Gravity compensation has been reported to induce plant

responses similar to the effects of microgravity on plant

development (193). Curtis (48) reported that pustules of

the bean rust, caused by Uromyces phaseoli (Pers.) Wint.,

developed faster on gravity compensated beans compared to

nonrotated control plants. Wells and Baker (232) reported










that the fresh and dry weights of tumors induced by

Aarobacterium tumefaciens (Smith & Towns) Conn. were

significantly larger on carrot disks rotated on a clinostat

compared to tumors on nonrotated controls. These results

support the hypothesis that the severity of disease may be

greater in a microgravity environment than in a normal

terrestrial gravity. Although Brown et al. (25) cautioned

that all plants do not behave on clinostats exactly as they

behave in microgravity, the effects of microgravity on plant

(46,87,121,155,156,202) and microbial (2,139,170,214,240)

physiology support the hypothesis that new and unique host-

pathogen interactions may occur in a space-based CELSS.

Further research in microgravity phytopathology is obviously

required to determine the possible deletirious effects of

microorganisms on plant health.

It seems apparent that, because hydroponic plant

production systems are proposed for space-based CELSS

(30,101,124,166,219), an integrated pest management (IPM)

program might be developed for CELSS by applying available

information from terrestrial hydroponic systems. An IPM

program for CELSS will likely include components of

quarantine, sanitation, compartmentalization, plant

resistance, and constructed microbial communities (158,159).

In addition, ultraviolet radiation (35,67,210), heat

treatment (191), ozonation (190), ultrafiltrtation

(190,199), and bacterization (4,223,224) have proven

partially to moderately effective in suppressing root










diseases in terrestrial hydroponic systems and may be

applicable to CELSS. However, limitations in efficacy,

phytotoxicity, applicability, and energy requirements may

reduce the utility of these physical control options in

CELSS. Chemical biocides also have been proposed as pest

control options for space-based CELSS (219) and have proven

effective in terrestrial hydroponic systems for the control

of root diseases caused by Fusarium spp. (145,209,221).

However, strict air and water quality protocols for American

spacecraft (18) appear to preclude their use in CELSS.

Environmental manipulation of CELSS modules has not

been emphasized previously as an IPM component for disease

control, but it seems ideally suited for hydroponic plant

production systems in CELSS. Components of CELSS plant

production systems will be engineered for maximum

environmental controllability and compartmentalization to

optimize horticultural conditions for specific crops (124)

and to contain disease outbreaks (124,166). Temperature has

been used effectively to control root infection by Pythium

aphanidermatum (Edson) Fitzp. and P. dissotocum Drechsler

(15,77) under hydroponic conditions. Furthermore, altering

the hydrogen ion concentration of the nutrient solution has

been reported to reduce the severity of disease caused by

Phytophthora cinnamomi Rands (19) on avocado and Pythium

debaryanum Hesse on lettuce (236).

Before initiating these experiments, the myriad of

possible pathosystems that could be studied in CELSS at










first appeared overwhelming. Indeed, if no studies have

been conducted on the effects of microgravity on host-

pathogen interactions, the study of any pathosystem might be

pertinent. However, it was decided that the selection of a

pathosystem should be based on the microbial contamination

reported from spacecraft, and that it should be based on the

occurrence of that microorganism as a significant pathogen

in terrestrial hydroponic systems. Pathogens in the genus

Fusarium fulfilled these two requirements. Fusarium spp.

have been isolated from Apollo spacecraft (96,180,181,218),

Shuttle payloads (144), and in experimental ground-based

CELSS (57,94,127). Furthermore, Fusarium spp. comprise the

second largest group of root pathogens in terrestrial

hydroponic systems (45,66,106,165,174,176,177,182,183,

184,212).

The first objective of this study was to test several

pathosystems under hydroponic conditions. The hosts were

chosen based on their selection as candidate plant species

for CELSS (101,219) and on their use in experiments in

microgravity (87,46,139,132). Fusarium solani (Mart.)

Appel. & Wr. f. sp. phaseoli (Burk.) Snyd. & Hans. and Vigna

radiata (L.) Wilczek were selected for subsequent

experiments because severe disease was observed in the

pathogenicity tests, and because pathogenesis appeared to be

influenced by temperature in a preliminary experiment (197).

The second objective of this study was to determine the

effects of temperature, hydrogen ion concentration,










humidity, and light quality on disease caused by F. solani

f. sp. phaseoli. These parameters are easily controllable

in closed systems (124,178,192) and might be used to manage

plant diseases in CELSS while avoiding the inclusion of

subsystems such as ultraviolet radiation, ozonation, or

ultrafiltration. Disease control using environmental

manipulation may have the additional advantage of being

adaptable to different plant production systems. In

contrast, subsystems like ultrafiltration or bacterization

might not be as widely applicable because of unique

constraints among different plant production systems. For

example, bacterization of plant roots growing in a

microporous tube system (56) might differ from bacterization

of roots in a NFT system because roots in the tube system,

unlike those in a NFT system, are partially exposed to air.

The third objective of this study was to determine the

effects of temperature and hydrogen ion concentration on the

attachment of macroconidia of F. solani f. sp. phaseoli to

roots of V. radiata. It was hypothesized that disease

control might be more effective by interfering with spore

attachment than by suppressing pathogenesis after plants

became infected.

The final objective of this study was to determine

whether spore attachment could be disrupted by the addition

of hapten sugars that were specific for plant lectins.

Lectins (phytoagglutinins) have been implicated in the

attachment of microorganisms to root surfaces (73,91,128,










129,130). Furthermore, a phytoagglutinin has been isolated

from hypocotyl cell walls (114,115,86), seeds (90), and

leaves (105) of V. radiata. Although no literature was

found that described phytoagglutinins from roots of V.

radiata, the presence of phytoagglutinins in roots of other

legumes (22,54,74,75,206) suggested that they may be

present. The activity of the phytoagglutinin from V.

radiata was shown to be sensitive to changes in hydrogen ion

concentration (114,115), and it seemed reasonable to suspect

that it might be involved with attachment of macroconidia of

F. solani f. sp. phaseoli.

In summary, these experiments were designed to

determine the effects of several environmental parameters on

disease caused by a pathogen that might pose a threat to

plant health in a space-based CELSS. Spore attachment by

the pathogen to roots was investigated because the process

was hypothesized to be affected by parameters of the

nutrient solution that are easily controlled in hydroponic

systems. Management of root diseases by altering the

environment in terrestrial hydroponic systems and in space-

based CELSS might be an inexpensive method for disease

control that could reduce construction costs of both

systems.













CHAPTER 2
EFFECTS OF CULTURE MEDIA ON MACROCONIDIUM MORPHOLOGY AND
PATHOGENICITY OF FUSARIUM SOLANI F. SP. PHASEOLI


Introduction
Cultural conditions can greatly affect colony

morphology, pigmentation, and sporulation of Fusarium spp.

on artificial media (69,149,160,168,204). Culture media

high in carbohydrates, such as potato dextrose agar (PDA),

can induce segmentation of cultures (160,230) or the

formation of patch mutants (149) with a corresponding

alteration, usually a decrease, in fungal pathogenicity or

virulence (7,31,148,160,168,203,230,231). Growth of

Fusarium spp. on PDA can also result in increased

variability of macroconidium morphology (160,168), but it is

unclear whether macroconidium morphology is linked to

differences of pathogenicity in the genus Fusarium. Natural

media were proposed by Snyder and Hansen (205) to reduce

these problems and enhance sporulation of fungi in culture.

Fisher et al. (69) and Nelson et al. (160) suggested

the use of carnation leaf agar (CLA) for the storage and

taxonomy of Fusarium spp. because of culture stability and

uniformity of macroconidium shape. However, CLA has been

employed rarely for the production of inocula of Fusarium

spp. intended for pathogenicity tests. A review of papers










published in Phytopathology and Plant Disease, during the

periods of 1969 to 1989 and 1980 to 1989, respectively,

involving pathogenicity tests with Fusarium spp., indicated

that PDA was used for inoculum production in 43% of 244

studies; other artificial media rich in carbohydrates were

used in 19% of the studies; natural media (excluding CLA) or

naturally infested soil were used in 15% of the studies; and

various liquid media were used in 20% of the studies.

Carnation leaf agar was utilized in only 3% (7 of 244) of

the studies.

The objectives of this research were 1) to determine

the relative quality and quantity of macroconidia,

microconidia, and chlamydospores produced by isolates of

Fusarium spp. on different culture media; 2) to

quantitatively determine morphological differences among

macroconidia of Fusarium solani (Mart.) Appel. & Wr. f. sp.

phaseoli (Burk.) produced on two forms of PDA and two

natural media; and 3) to determine if there was variability

in pathogenicity or virulence of inoculum of E. solani f.

sp. phaseoli produced on different media. Portions of this

study were presented previously (197).


Materials and Methods

The identifications of Fusarium spp. were confirmed

with monoconidium cultures using the key of Nelson et al.

(83). The identification of F. solani f. sp. phaseoli was

based on pathogenicity tests (197) and on the key of Matuo










and Snyder (140). Cultures of Fusarium spp. were maintained

in long-term storage at 4.0 C in a lyophilized form or on

slants of modified carnation leaf agar (MCLA), which is

described below. Short-term storage for specific

experiments was on MCLA.

Sterilization of leaves of carnation (Dianthus

caryophillus L., cv. 'Improved White Sim') and stem pieces

of mung bean (Vigna radiata (L.) Wilczek, cv. 'Berken') was

similar to the procedure of Fisher et al. (69). Plant

material was placed in capped, 250-ml glass jars and

irradiated with 15.1 megarads of gamma irradiation using a

Cobalt-60 source (Dept. of Chemistry, University of Florida,

Gainesville, FL, 32611). Irradiated samples were tested for

sterility by placing 30 randomly selected pieces of plant

material from each treated container on nutrient agar

composed of 3 g beef extract, 2.5 g glucose, 5 g peptone,

and 15 g of Bacto agar (Difco Laboratories, Detroit, MI,

48232) per liter of deionized water. Thirty pieces also

were placed on potato dextrose agar (Difco Laboratories)

supplemented with 1.0 ml of Tergitol NP-10 (Sigma Chemical

Co., St. Louis, MO, 63178) and 50.0 mg of chlortetracycline

hydrochloride (Sigma Chemical Co, St. Louis, MO, 63178)

(PDATC) per liter of medium (137).

Carnation leaf agar, prepared as was described by

Nelson et al. (160), consisted of irradiated carnation leaf

pieces placed on 2% water agar. Modified carnation leaf

agar was prepared by placing five to eight pieces










(approximately 3 mm x 5 mm) of irradiated plant material on

a nutrient solution agar (NSA) containing 2% Bacto agar and

the following inorganic salts (all obtained from Sigma

Chemical Co.): 4.51 mM Ca(N03)2.4H20, 3.48 mM KNO3, 1.00 mM

KH2PO4, 1.65 mM MgSO4*7H20, 0.037 mM H3BO4, 7.28 AM

MnSO4.H20, 4.59 AM ZnSO4-7H20, 3.05 MM CuC12-2H20, 0.074 pM

(NH4)6 Mo7024*4H20, and iron provided by equimolar

concentrations (89.5 AM) of FeCl3*6H20 and diethylene-

triaminepentaacetic. Hydroponic nutrient solutions used for

the following experiments were mixed fresh with

concentrations of ions identical to those used for NSA.

Potato dextrose agar was prepared as described by the

manufacturer (Difco Laboratories).

Forty-three isolates of Fusarium spp. (Table 2-1) were

evaluated for production of macroconidia, microconidia, and

chlamydospores on PDA, CLA, and MCLA. Macroconidia of

Fusarium spp. from MCLA cultures were streaked onto freshly

prepared PDA, CLA, or MCLA. Cultures were incubated for

14 days at 25 C under a 12-hr photoperiod. Light was

supplied by two, 20-watt, fluorescent bulbs at an intensity

of 30 Amol.m-2.s-1. Light intensity was measured with a

solar-IR light sensor (Campbell Scientific, Inc., Logan, UT,

84321) attached to an Omnidata Polycorder (Omnidata

International, Inc., Logan, UT, 84321). Cultures were first

studied without disturbing the agar surface using a stereo

microscope. Conidia, conidiophores, and chlamydospores were

then removed from each medium and stained in 0.05% trypan

















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blue (Eastman Kodak, Rochester, NY, 14650) in a lactic

acid:water:glycerol solvent (1:1:2). Further observations

were conducted under bright-field illumination with a Nikon

Optiphot compound microscope.

Pathogenicity tests were conducted on 19 pathosystems

with different cultivars of Phaseolus vulaaris L., Pisum

sativum L., Triticum avestivum L., and V. radiata

(Table 2-2). Seeds from each plant cultivar were germinated

in rolls of seed germination paper, as was described by

McClure and Robertson (141). Seven- to ten-day-old plants

of each cultivar were placed individually into five, 250-ml

flasks containing 225 ml of nutrient solution per flask and

grown under four, 40-watt, standard fluorescent bulbs at an

intensity of 100 Amol*m-2.s-1. Inoculum was prepared from

14-day-old MCLA cultures. Plants were inoculated at varying

densities between 350 and 8000 propagules/ml of nutrient

solution. The pH and temperature of the nutrient solution

were, respectively, 6.0-6.5 and 23-25 C at the time of

inoculation. Plants were maintained at room temperature

(approximately 23-25 C) for 14 days and then inspected for

root symptoms and wilt. Compressed air was passed through a

0.45-Am cartridge filter (Gelman Versaflow filter, Gelman

Sciences, Inc., Ann Arbor, MI, 48106) and distributed to

each 250-ml flask to aerate the nutrient solution.

Four pathosystems using E. solani f. sp. phaseoli

(Table 2-2) were selected for tests at 20, 25, and 30 C to

determine the effect of temperature on disease.









Table 2-2. Fusarium spp. tested for pathogenicity on
various hosts in a hydroponic system.

Isolate
Fungus designation Host


F. solani f. sp.
phaseoli


F-28AV


Phaseolus vulgaris
cv. Kentucky Wonder
cv. Pinto
cv. Red Kidney


Vi.na radiata
cv. Berken


F. solani f. sp.
pisi


W8W Pisum sativum
cv. Alaska
cv. Freezonian
cv. Tom Laxton


. graminearum


F. culmorum



E. oxysporum


E. oxysporum
f. sp. pisi


R2D-10-7X


6A72AX



F-5Y


Reiling-R2z


Triticum avestivum
cv. Olsen's Dwarf
cv. Urquie
cv. Wampum
cv. Waverly
cv. Yecorro Rojo

T. avestivum
cv. Olsen's Dwarf
cv. Yecorro Rojo

T. avestivum
cv. Olsen's Dwarf
cv. Yecorro Rojo

P. sativum
cv. Alaska
cv. Freezonian
cv. Tom Laxton


V Obtained from A. J. Anderson, Utah State University,
Logan, UT, 84322; isolate designation assigned at The
Land, EPCOT Center, Lake Buena Vista, FL 32830.
SObtained from J. M. Kraft, United States Department of
Agriculture, Agriculture Research Service, Prosser, WA,
99350.
x Obtained from R. J. Cook, Washington State University,
Pullman, WA, 99163.
y Obtained from The Land greenhouses, EPCOT Center, Lake
Buena Vista, FL 32830.
z Obtained from J. J. Marois, University of California,
Davis, CA 95616.










Plants were grown in EGC model M-13 growth chambers

(Environmental Growth Chambers, Chagrin Falls, Ohio, 44022)

under conditions similar to those of the pathogenicity

tests, except that the photon flux was raised to 250-275

nmolm-2.*s-1. Seven plants per cultivar were used at each

temperature per each experiment. Inoculum of the pathogen

was prepared from a single isolate (F-28A) grown on MCLA for

14 days at 23-25 C. Plants were inoculated at a density of

500 propagules/ml. After 14 days plants were harvested and

the fresh and dry weights determined for each plant. The

experiment was conducted three times. A completely

randomized design was used to position replicates of each

treatment within growth chambers. The pathosystem of E.

solani f. sp. phaseoli and V. radiata, cv. 'Berken,' was

selected for further experiments because plant disease was

responsive to temperature changes and other pathosystems did

not consistently develop disease.

In a separate series of experiments, four different

media were used to produce macroconidia of F. solani f. sp.

phaseoli for studies on spore morphology and plant

pathogenicity. Preparation of PDA and MCLA was described

above. Fresh potato dextrose agar (FPDA), prepared as was

described by Nelson et al. (160), consisted of a potato

broth solidified using 1.5% Bacto agar; 2.0% dextrose was

added to the unsolidified potato broth. Mung bean stem agar

(MBSA) was prepared by placing 10-12, 1-cm-long pieces of

gamma-irradiated mung bean stems (V. radiata, cv. 'Berken')










on NSA. Macroconidia were streaked onto freshly prepared

MCLA, MBSA, PDA, and FPDA.

To determine the morphological differences among

macroconidia of F. solani f. sp. phaseoli produced on the

four different media, macroconidia were separately collected

from each medium using a platinum-wire loop and submersed in

20.0 p1 of 0.05% trypan blue stain for 5 min. Three

microliters of the stained macroconidium suspensions were

transferred to NSA and glass coverslips placed over the

microdrops. Agar blocks with the spores and coverslips were

excised from petri dishes and placed on glass microscope

slides. Macroconidia were observed using a Nikon Optiphot

microscope electronically coupled to a MicroComp* Integrated

Video Image Analysis System (Southern Micro Instruments,

Inc., Atlanta, GA, 30339). The length and width of 30,

randomly selected macroconidia from each sample were

measured. Five spore samples were prepared from separate

cultures in each of three experiments. In preliminary

tests, the accuracy of repeated measurements using the

MicroComp Integrated Video Image Analysis System was

determined to be within 2% of the mean. The length of each

macroconidium was measured as depicted in Figure 2-1A. The

width of each macroconidium was measured at the widest point

of the spore. The procedure of mounting the stained

suspension of conidia on an agar surface was essential for

accurate measurements. Macroconidia would come to rest on

their lateral sides permitting precise measurement of each
































Figure 2-1. Comparison of macroconidia of Fusarium solani
f. sp. phaseoli mounted on nutrient solution agar and in a
lactic acid, water, and glycerol solvent. A, Macroconidia
mounted in 0.05% trypan blue stain on nutrient solution agar
were measured in short straight lines to accommodate the
camber of the spore. B, Macroconidia mounted in trypan blue
stain or solvent were not accurately measured due to severe
differences in the focal planes between portions of
individual conidia.






24










conidium. When macroconidia were mounted on glass slides in

trypan blue stain or clear solvent, they were randomly

distributed at various angles (Figure 2-1B); individual

macroconidia would often fail to settle within a single

focal plane, and, thus, were very difficult to measure

accurately. Photographs were taken using Polaroid* 4x5

Instant Film, Type N55 (Polaroid Corp., Cambridge, MA,

02139). Data were analyzed using the Statistical Analysis

System (SAS) (SAS Institute, Inc., Cary, NC, 27512).

Treatments were compared for equality in an Analysis of

Variance followed by Fischer's Least Significant Difference

Test (169) (P = 0.05).

A hydroponic system was used to test the pathogenicity

and virulence of inocula of F. solani f. sp. phaseoli

produced on PDA, FPDA, MCLA, and MBSA. Root temperature was

controlled at 20.0 0.5 C using a closed-loop tube system

connected to a Lauda RMS-20, chilled-water, recirculating

bath (Brinkman Instruments, Inc., Westbury, NY, 11590). A

single, 30-cm-long coil of 3.5-mm-diameter, stainless steel

tubing was placed into each plastic container and connected

to the main reservoir of the water bath by clear plastic

tubing (NalgeneM 8000 tubing, Nalgene Co., Rochester, NY,

14602). Experimental units consisted of 4-liter, plastic

containers wrapped on the sides with closed-celled foam,

duct-insulation tape (Macklenburg-Duncan, Oklahoma City, OK,

73118). Light was excluded from the nutrient solution by

the foam wrapping on the sides of the plastic containers and










by foam or black tape on the upper surface of the container

lids. The pH of the nutrient solution in each plastic

container was adjusted to 6.0 0.2 units every 24 hr using

0.01 N HNO3 and 0.02 N KOH. A completely randomized design

was used to assign treatments to experimental units.

The following methods were tested for their utility in

producing seedlings of V. radiata: a slant-board culture

method (116), a method using rolls of seed germination paper

(141), a growth pouch method (31), and germination of seed

in 20-mesh silica sand or vermiculite. Problems were

encountered with these procedures that reduced the quality

of seedlings. Root hairs adhered to seed germination paper

in the first three procedures, resulting in root damage when

plants were transplanted. Branching characteristics of V.

radiata in all five methods were not similar to root

branching characteristics observed in hydroponic solutions.

A new technique was developed that minimized root contact

with solid materials and optimized root contact with the

hydroponic solution.

Seed of V. radiata, cv. 'Berken,' were surface steril-

ized for 15 min in 0.13% NaOCl (2.5% bleach, Kare Chemical

Co, Opalocka, FL, 33054) plus 1.0 ml of Tween-20 (J.T. Baker

Chemical Co., Phillipsburg, NJ, 08865) per liter of

solution. Seed were rinsed three times in sterile deionized

water, allowed to imbibe water for 12 to 18 hr, and sown in

a seed germination tray (Figure 2-2) modified from a system

described by Prince and Knott (178). Plant supports of the







































Figure 2-2. Schematic drawing of a cross-section through a
seed germination tray (t) used for the production of
seedlings of Vigna radiata. Extruded plastic struts (ps)
were assembled by joining overlapping sections with two-part
epoxy (ep). A single layer of fiber-reinforced plastic
sheeting (p) was cut to fit through gaps between plastic
struts and extended low enough to be submersed 1.0-1.5 cm
below the surface of the nutrient solution (ns). Seed
germination paper (gp) was cut and fit over the plastic
sheeting to function as a wick for the nutrient solution.
Seed (s) were placed with their hilums down between opposing
surfaces of adjacent sections of seed germination paper.
Saran wrap (sw) was placed over the seed germination tray
for 3 days to enhance seed germination and hypocotyl (h)
extension. Cotyledons (c) would often dislodge from the
stem before transplanting seedlings. Root (r) growth
consisted primarily of first- and second-order roots.










seed germination trays were constructed of extruded plastic

struts, which were assembled in a series of slats using

common two-part epoxy. A single layer of fiber-reinforced

plastic sheeting was cut to fit through the gaps between the

plastic struts and then extended low enough to be submersed

1.0-1.5 cm below the surface of the nutrient solution. Seed

germination paper (no. 38 seed germination paper, regular

weight, Anchor Paper, St. Paul, MN 55101) was cut and fit

over the plastic sheeting to function as a wick for the

nutrient solution. Seeds were placed with their hilums down

between opposing sheets of seed germination paper (Figure

2-2). Plant supports were constructed to fit snugly into

28- x 38-cm polyethylene trays. Each tray was filled with

7.0 L of nutrient solution at pH 6.0-6.5.

Seed germination trays were covered with a single layer

of plastic saran wrap (Sealwrap, Borden Chemical, North

Andover, MA, 01845) to enhance seed germination and

hypocotyl extension, and placed under a combination of two,

400-watt, high pressure sodium lamps and two, 250-watt,

metal halide lamps (Energy Technics, York, PA, 17402).

Photon flux was 300 pmol*m-2*s-1 at the top of seed

germination trays and was supplied in a 14-hr photoperiod.

The saran wrap was removed after 3 days and seedlings were

allowed to develop an additional 4-5 days. Root growth was

uninhibited and consisted of first- and second-order roots

(70).










Five seedlings were transplanted into each plastic

container filled with 4.0 L of freshly mixed nutrient

solution. Each seedling was held in place by securing its

stem with a 28-mm-diameter, open-celled, foam plug (Dispo'

Plug, Baxter Healthcare Corporation, Stone Mountain, GA,

30083). Since a previous report suggested that open-celled

polyurethane foams can release phytotoxic volatiles and

should be heat or chemically treated prior to use (233),

foam plugs were heated at 70 C for 48-96 hr and then

autoclaved at 121 C and 1.1 kg.cm-2 for 25 min prior to use.

The nutrient solution in each plastic container was

aerated with filter-sterilized air using a 0.45-Mm cartridge

filter (Gelman Versaflow filters, Gelman Sciences, Inc., Ann

Arbor, MI, 48106) to maintain dissolved oxygen near

saturation (9.0-9.2 mg.L-1 at 20 C). Plants were grown

under eight, 1500-milliamp, Power Groove* fluorescent lamps

(General Electric Co., Cleveland, OH, 44112) which supplied

250-275 jmol*m-2.s-I at the top of the plant canopies.

The seed germination trays and experimental equipment

were assembled in a 2.5-m x 4.5-m research laboratory.

Ambient temperature was controlled by thermostatically

adjusting room temperature. Ambient humidity was provided

by a Through-Wall Humidifier (Model 707-TW, Hermidifier,

Inc., Lancaster, PA, 17603). Ambient temperature and

humidity for these tests were 21.3 1.5 C and 60.9% 8.0%

relative humidity, respectively. Nutrient solution tempera-

ture, ambient temperature, and ambient humidity data were










collected using the Campbell Scientific CR7X Measurement and

Control System (Campbell Scientific, Inc., Logan, UT, 84321)

and processed using The Land's Database Management System,

as has been described previously (157).

Inocula were prepared from 14-day-old cultures of PDA,

FPDA, MBSA, and MCLA. Plants were inoculated by adding

50 ml of a macroconidium suspension to produce a final spore

density of 500 propagules/ml of nutrient solution. Plants

were allowed to equilibrate in the plastic containers for

24 hr prior to inoculation. Two replicates per treatment

were inoculated in each experiment; two noninoculated

replicates served as controls. A completely randomized

design was used to assign treatments to experimental units.

The experiment was conducted three times. Fresh weights and

dry weights of individual plants were measured 14 days after

inoculation. The mean plant weight per experimental unit

was calculated, and data were subjected to an Analysis of

Variance to test for overall treatment equality, and

subsequently to Fischer's Least Significant Difference tests

(169) (P o 0.05).


Results
Isolates of the Fusarium spp. tested varied in

appearance between pinnotal forms and mycelial forms as has

been described by Nelson et al. (160). Patch mutants (149)

were observed on PDA with all isolates of F. solani,

E. culmorum (Smith) Sacc., E. graminearum (Schwabe) Snyd. &










Hans., and F. lateritium Nees emend. Snyd. & Hans., but were

observed less frequently with isolates of F. oxVsporum

(Schlect) Snyd. & Hans. Patch mutants occurred rarely on

MCLA and CLA. Sporodochia and phialides were present on the

wild-type mycelium of most isolates, but they were lacking

in patch mutants. Most isolates of Fusarium spp. produced

aberrant macroconidia and reduced quantities of

chlamydospores on PDA, as compared to the formation of these

structures on MCLA and CLA (Table 2-1). Aerial mycelium was

suppressed in most cultures grown on MCLA, when compared to

mycelium on CLA. Production of chlamydospores was

dramatically greater on both MCLA and CLA in cultures with

agar surfaces disturbed, such as by incisions from a

scalpel, than in undisturbed cultures. Macroconidia were

primarily produced on carnation leaf pieces on MCLA and CLA,

while chlamydospores were observed exclusively below the

agar surface.

In the preliminary pathogenicity tests, symptoms failed

to develop on T. avestivum inoculated with F. culmorum and

E. oxysporum, even at the highest inoculum density of 8000

propagules/ml of nutrient solution. Root necrosis was

observed on all of the wheat cultivars when plants were

inoculated with F. graminearum at 500 propagules/ml;

greatest disease occurred on cultivars Wampum and Urquie.

However, plant wilt did not occur with any of the wheat

cultivars inoculated with F. graminearum, at any inoculum










level, nor was there any dramatic difference in vegetative

growth between inoculated and noninoculated plants.

Root symptoms developed on the pea cultivar Alaska, but

only when the inoculum density of F. solani f. sp. Disi

(Jones) Snyd. & Hans. exceeded 5000 propagules/ml. Fusarium

oxvsporum f. sp. pisi (Van Hall) Snyd. & Hans. induced root

symptoms on all three cultivars of E. sativum, but the

cultivar Freezonian was the least resistant. Plant wilt did

not occur in any pea cultivar inoculated with F. oxysporum

f. sp. pisi.

Bean cultivars inoculated with F. solani f. sp.

phaseoli developed severe root necrosis that resulted in

either plant wilt or reductions in vegetative growth of the

host. In subsequent studies on the effects of temperature

on disease, 20-52% of P. vulgaris or V. radiata plants

wilted between 10 and 14 days after inoculation at 20 C.

Greatest plant damage occurred with the mung bean cultivar

Berken; 52% of plants wilted at 20 C and 21% of the plants

wilted at 25 C. Plant wilt did not occur at 30 C with any

of the bean cultivars. Further studies on the effects of

temperature on disease caused by F. solani f. sp. phaseoli

in V. radiata are presented in Chapter 3.

Distinct morphological differences were observed among

macroconidia produced on the different media. Macroconidia

produced on MCLA and MBSA (Figures 2-3A and 2-3B) stained

darker with trypan blue and had greater internal detail of

the cells than macroconidia produced on PDA and FPDA
































Figure 2-3. Comparison of macroconidia of Fusarium solani f.
sp. phaseoli macroconidia produced on four different culture
media. Macroconidia were grown, harvested, stained, and
photographed, and negatives were printed, in identical
manners; thus, differences in size, cell-content complexity,
staining qualities, septation, and spore uniformity are
comparable. The dimension bar is applicable to all images.
Macroconidia produced on modified carnation leaf agar (MCLA)
(A) stained darker than those in other treatments.
Macroconidia produced on mung bean stem agar (MBSA) (B) were
similar to macroconidia produced on MCLA. Macroconidia
produced on MCLA and MBSA showed greater detail in their
cell contents than macroconidia produced on Difco potato
dextrose agar (PDA) (C) or fresh potato dextrose agar (FPDA)
(D). Macroconidia produced on PDA and FPDA were similar to
each other; they exhibited swollen and distended terminal
and intercalary cells, showed reduced staining
characteristics compared to macroconidia grown on MCLA and
MBSA, and had increased nonuniformity of conidium walls.






34










(Figures 2-3C and 2-3D). Septa were sharply delineated and

thicker in macroconidia produced on MCLA and MBSA, whereas

septa were not easily observed in macroconidia produced on

PDA and FPDA. Nonuniform and misshapen macroconidia were

produced in cultures of PDA and FPDA. Terminal and foot

cells of macroconidia were periodically distended (Figures

2-3C and 2-3D); swollen intercalary cells of macroconidia

were also observed. Severe distortions occurred on a very

low percentage of macroconidia produced on PDA and FPDA.

The predominant morphological distortion present on

macroconidia produced on PDA and FPDA was nonuniformity of

the side-walls of each conidium. Misshapen or distorted

macroconidia were not observed in MCLA or MBSA cultures.

The lengths and widths of macroconidia produced on all

media were distinctly different from each other (Table 2-3).

Macroconidia produced on PDA and FPDA were shorter and wider

than macroconidia grown on MCLA and MBSA (P < 0.05).

Furthermore, the variability of length and width was greater

for macroconidia produced on PDA and FPDA than for those

produced on MCLA and MBSA.

In the present study, distinct morphological

differences were observed in macroconidia produced on

different media. However, no differences in pathogenicity

or virulence were observed in tests with the pathosystem of

E. solani f. sp. phaseoli and V. radiata using macroconidia

produced on PDA, FPDA, MCLA, and MBSA (Table 2-4). Fresh









Table 2-3. The length and width of macroconidia of Fusarium
solani f. sp. phaseoli grown on four different media.


Media Length (Am) Width (im)

Mean Range Mean Range


Potato dextrose
agar 52.5 az 31.0-70.5 5.0 a 3.1-9.2

Fresh potato
dextrose agar 56.0 b 37.8-75.0 4.7 b 3.1-8.0

Modified carnation
leaf agar 59.8 c 49.8-75.6 4.6 c 3.2-5.9

Mung bean stem
agar 61.9 d 49.1-74.4 4.6 c 3.2-6.0


Z Table values represent the means of 15 replicates (five
replicates per each of three experiments). Data were
analyzed using an Analysis of Variances followed by
Fischer's Least Significance Difference test. Values
followed by the same letter were not different (P > 0.05).









Table 2-4. Effects of culture media on the virulence of
inoculum of Fusarium solani f. sp. Dhaseoli on disease in
Viqna radiata.

Plant Plant Percent
fresh weight dry weight plant
Treatment (g) (g) wiltY

Noninoculated
control 4.88 aY 0.42 a 0 az

Potato
dextrose agar 1.54 b 0.18 b 83 b

Fresh potato
dextrose agar 1.79 b 0.22 b 67 b

Modified
carnation
leaf agar 1.77 b 0.21 b 77 b

Mung bean
stem agar 1.51 b 0.18 b 83 b


Y Table values represent means of six replicates (two
replicates per each of three experiments). Data were
analyzed using Analysis of Variance and Fisher's Least
Significant Difference test; values followed by the same
letter were not different (P > 0.05).
z Data presented as percentages of plant wilt were
transformed to arcsines of square roots and then subjected
to an Analysis of Variance test followed by Fisher's Least
Significant Difference test; values followed by the same
letter were not different (P > 0.05).










weights and dry weights of plants receiving inoculum

produced on the different media were not different from each

other, but were different from noninoculated control plants

(P 5 0.05). Plant wilt was also similar among the

treatments receiving inoculum.


Discussion

Macroconidium and colony morphology of Fusarium spp.

can be altered by ultra-violet light (148), differences in

wide-spectrum light quality (204), and cultural conditions

(69,148,149,160,168,194). Results of the study with 43

isolates of Fusarium spp. grown on PDA, CLA, and MCLA

indicate that variations in macroconidium morphology

produced on PDA occurred in a wide range of species in the

genus Fusarium. Variations in length and width, and the

development of swollen intercalary cells, were observed in

macroconidia produced on PDA. In contrast, macroconidia

produced on MCLA and CLA exhibited few distortions and were

of uniform size. In addition, the relative abundance of

macroconidia, microconidia, and chlamydospores from Fusarium

spp. grown on PDA, MCLA, and CLA were different. The width

and length of macroconidia, of at least a few Fusarium spp.,

have been shown to be altered by production on media with

high carbohydrate concentrations (69,136,168,194). Marchant

and White (136) reported swollen intercalary cells in

macroconidia of E. culmorum that were grown in the presence

of glucose and a nitrogen source. Macroconidia of E. solani










f. sp. phaseoli also developed swollen intercalary cells

when they were incubated in 50-mM solutions of glucose,

sucrose, and mannose (Chapter 6). Spore swelling was not

observed when macroconidia were allowed to germinate on root

surfaces (Chapter 4). Thus, it appears that morphometric

changes of macroconidia may represent artifacts induced by

the production or incubation of spores in environments high

in carbohydrates.

Morphological characteristics of macroconidia are

critical for current taxonomic systems in the genus Fusarium

(20,160,218), and the need for standardization of cultural

conditions for taxonomic purposes (160) is generally

accepted. However, the usefulness of macroconidium

morphology for predicting the pathogenicity of cultures of

Fusarium spp. is questionable. Although previous workers

have demonstrated that cultural variability of Fusarium spp.

induced on media high in carbohydrates can correlate to

alterations in pathogenicity (7,69,148,160,194,230,231), few

studies have concomitantly investigated morphological and

pathological differences between macroconidia produced on

different media (80,194,230,231). Oswald (168) identified

spore width as the primary difference among culture variants

of F. culmorum, E. equiseti (Corda) Sacc., and F.

graminearum, but did not correlate changes in spore

morphology to changes in pathogenicity. Sano and Ui (194)

reported that pathogenicity and macroconidium length were

less, and macroconidium width greater, when inoculum of E.









solani f. sp. phaseoli was produced on a medium high in

carbon. Morphometric differences in the length and width of

macroconidia of F. solani f. sp. phaseoli produced on the

high carbon-containing media PDA and FPDA were consistent

with the study of Sano and Ui (194), but in the current

study, no differences were observed in pathogenicity with

inoculum produced on MCLA, MBSA, PDA, and FPDA.

The severity of disease caused by E. solani f. sp.

Dhaseoli in V. radiata (Chapter 3) and spore attachment of
the pathogen to roots of the host (Chapter 4) have been

shown to be affected by temperature and hydrogen ion

concentration of the nutrient solution. It has yet to be

demonstrated whether these environmental factors alter the

virulence of Fusarium spp. grown on different media.

Differences between the results from the current study and

those of Sano and Ui (194) might be due to different plant

production methods, culture media utilized for the

production of inoculum, or temperature and hydrogen ion

effects on spore attachment or virulence of inoculum.

It is not clear whether Sano and Ui (194) maintained

cultures of F. solani f. sp. phaseoli by repetitive

transfers on media with a high carbon concentration.

Previous studies have shown that repetitive transfers on

high carbon-containing media could induce increased

morphological variability of macroconidia (69,160,168,194)

and alter the pathogenicity of E. solani f. sp. phaseoli

(148,160,168,203). In the present study, inoculum was










prepared by transferring spores from cultures of MCLA, a low

carbon-containing medium, to media with either high or low

carbon; the effects of repetitive transfers with each medium

on pathogenicity were not evaluated.

Although it is unclear whether macroconidium morphology

is linked to differences in pathogenicity in the genus

Fusarium, culture variability, notably between the pinnotal

colony type and mycelial colony type, has been correlated to

modifications in pathogenicity (69,160,168,230,231).

Wellman and Blaisdell (230,231) described five basic colony

forms produced by E. oxvsporum f. sp. Ivcopersici (Sacc.) on

different media that correlated to consistent differences in

pathogenicity. Results from the current study do not

support the conclusion that macroconidium variability

correlates to differences in fungal pathogenicity.

Additional research is required before any comprehensive

conclusions can be drawn on the concomitant effects of

culture media on spore morphology and fungal pathogenicity

in the genus Fusarium.














CHAPTER 3
EFFECTS OF TEMPERATURE, HYDROGEN ION CONCENTRATION,
HUMIDITY, AND LIGHT QUALITY ON DISEASE CAUSED BY FUSARIUM
SOLANI F. SP. PHASEOLI IN VIGNA RADIATA


Introduction

Controlled ecological life support systems (CELSS) have

been proposed for space bases to regenerate oxygen, water,

and food from human and industrial wastes (18,133,166).

Biological processes will be combined with physical and

chemical technologies to provide life support systems that

will reduce requirements for the resupply of materials from

Earth. Higher plants are considered the primary candidates

for photosynthesis in CELSS (101,166,178,219) and various

methods of plant production have been proposed

(30,56,166,178,235). Hydroponic plant production systems

appear to have several advantages over lunar regolith (30)

and other solid substrates (166).

Plants in CELSS may be threatened by microorganisms

transported into space as contaminants on spacecraft

components, equipment, astronauts, and plant-propagative

materials. Extensive microbial contamination has been

reported in American spacecraft (24,96,131,144,180,181,

215,217,218,228). Species of Alternaria, Asperaillus,

Candida, Cephalosporium, Cladosporium, Fusarium, Mucor,

Penicillium, Phoma, Saccharomvces, and Trichoderma were









among the most prevalent fungal contaminants of these

systems. Predicting deletirious plant-microbe interactions

in space-based CELSS is hampered by an extreme paucity of

information on plant-microbe interactions in space.

However, because hydroponic plant production systems will be

utilized in space-based CELSS (30,101,124,166,219), it seems

likely that an integrated pest management (IPM) program

might be developed by applying available information from

terrestrial hydroponic systems.

In terrestrial environments, various plant pathogens

have been described as significant problems in hydroponic

systems. Species of Pvthium and Phvtophthora have been the

most frequently reported root pathogens in terrestrial

hydroponic systems (15,29,66,72,77,100,117,175,198,208,210,

211,225). However, species of Pythium and Phvtophthora have

not yet been detected as contaminants in American spacecraft

and should be avoidable in space-based CELSS through strict

sanitation and quarantine procedures.

Fusarium spp. have been isolated from Apollo spacecraft

(96,180,181,218), shuttle payloads (144), and in

experimental ground-based CELSS (57,94,127). Furthermore,

Fusarium spp. comprise the second largest group of root

pathogens in terrestrial hydroponic systems (45,66,106,165,

174,176,177,182,183,184,212,226). Couteaudier and

Alabouvette (page 153, cit. 45) concluded that wilt diseases

caused by Fusarium spp. were ". .the most important

pathological problem in soilless culture." Nowicki (165)









found that formae specials of Fusarium oxsporum

(Schlecht.) and Fusarium solani (Mart.) Appel. & Wr.

constituted 51% (213 of 415 isolates) of seven Fusarium spp.

identified as pathogens of greenhouse-grown cucumbers during

a 5-year survey in Poland. Based on this literature, it

seems apparent that Fusarium spp. will likely occur as

contaminants in space-based CELSS, and when present they may

constitute a threat to plants.

An integrated plant disease control program for CELSS

will likely include components of quarantine, sanitation,

compartmentalization, plant resistance, and constructed

microbial communities (158,159). Environmental manipulation

of CELSS modules has not been emphasized previously as an

IPM component for disease control, but seems ideally suited

for hydroponic plant production systems in CELSS. Although

temperature has been used effectively to control root

infection by Pvthium aphanidermatum (Edson) Fitzp. and P.

dissotocum Drechsler under hydroponic conditions (15,77),

few other studies have been found that explore the area of

environmental manipulation for disease control in hydroponic

systems (95,117,207).

The objectives of this study were 1) to test the

effects of temperature, hydrogen ion concentration,

humidity, and light quality on disease caused by F. solani

f. sp. phaseoli on Viqna radiata (L.) Wilczek under

hydroponic conditions; 2) to determine the fluctuations in

nutrient solutions of primary and secondary inoculum of










E. solani f. sp. phaseoli under different environmental

conditions; 3) to assess whether plant growth-inhibiting or

growth-promoting compounds accumulated in nutrient solutions

during experiments on the environmental effects on disease;

and 4) to determine if severe root disease contributes to

yield reductions in V. radiata by inducing plant nutrient

deficiencies. Vigna radiate was chosen for these studies

based on its selection as a candidate crop species for CELSS

(219) and its sensitivity to E. solani f. sp. phaseoli under

hydroponic conditions (Chapter 2).


Materials and Methods

Experiments on temperature, hydrogen ion concentration

([H+]), and humidity were conducted in environmental plant-

growth chambers (Model M-13, Environmental Growth Chambers,

Inc., Chagrin Falls, OH 44022). Light for each growth

chamber was provided in a 12-hr diurnal cycle by 16, 1500-

milliamp, Power-Groove fluorescent lamps (General Electric

Co., Cleveland, OH, 44112) and six, 25-watt, incandescent

bulbs. Florescent lamps were periodically changed, and

distances between the lights and plant canopies were

periodically adjusted to maintain a constant photon flux of

250 gmol.s- *m-2 at the tops of the plant canopies. Light

intensity for all experiments was measured with a Li-Cor,

LI-190SB, radiation sensor (Li-Cor, Ltd., Lincoln, NE,

68504) calibrated to measure spectral radiance between 400

and 700 nm.









Experiments on light quality were conducted in a 2.5-m

x 4.5-m, tissue culture clean-room. High intensity

discharge (HID) lamps were suspended over the plant-growing

surface from metal supports made of 2.54-cm aluminum tubing.

To prevent cross-exposure to light, HID lamps were separated

by suspending sheets of white plastic from the metal

supports. Complete light spectra of HID lamps were obtained

using a Li-Cor, LI-1800 spectroradiometer (Li-Cor, Ltd.,

Lincoln, NE 68504) calibrated to determine lamp output in 2-

nm bands between 330 and 1100 nm. Light was provided in a

12-hr diurnal cycle by the following HID lamps: two, 250-

watt, high pressure sodium (HPS) fixtures (Energy Technics,

York, PA, 17402); two, 400-watt, metal halide (MH) fixtures

(Energy Technics, York, PA, 17402); and eight, high

intensity fluorescent bulbs (FL) (Chapter 2) assembled in a

specially designed 91.4-cm x 121.9-cm fixture (New Horizons

Lighting, Palm City, FL, 34990). Light intensity from each

combination of lamps was measured weekly; as lamps aged

light intensity would drop by up to 20% with FL lamps and

between 5-10% with MH and HPS lamps. The distances from the

HID lamps to plant canopies were periodically adjusted to

maintain a constant photon flux of 250 pmol.s-l*m-2 at the

tops of the plant canopies. Fluorescent lamps were changed

every 4 to 8 weeks to assist in maintaining the appropriate

light intensity. Plexiglass barriers (Rohm & Haas,

Philadelphia, PA, 19105) were placed between all lamp









fixtures and plant canopies to reduce radiant and convective

heating of plant foliage.

Air circulation was provided in each HID lamp zone by

individual air blowers (Model 4C446 Dayton Electric Mfg.

Co., Chicago, IL 60648) connected to the outlet of the room

air conditioning system. Air temperature was maintained at

20 or 25 C, 2.0 C, by adjusting the room thermostat. Root

temperature control was within 0.5 C of set-points and was

provided by Lauda temperature-control baths, as was

described previously (Chapter 2). Humidity was maintained

at a vapor pressure deficit (VPD) of 6.0 mm Hg using

individual humidifiers (Model 707-TW, Hermidifier, Inc.,

Lancaster, PA, 17603) in each HID lamp zone.

The following procedure for production of seedlings,

assembly of experimental units, inoculation of appropriate

treatments, and completion of each experimental repetition

was identical for all experiments and was similar to the

procedure described in Chapter 2. Experimental units

consisted of 4-L plastic containers wrapped in 5.0 mm of

foam insulation tape (Chapter 2). Eight plastic containers

were arranged in each HID lamp-zone or growth chamber.

Five, 7- to 8-day-old seedlings of V. radiata, cv. 'Berken,'

were transplanted into each plastic container; plants were

held in place with the aid of 28-mm-diameter, foam plugs

pretreated by dry heat and autoclaved, as was described

previously (Chapter 2). A modified Hoagland's nutrient

solution (Chapter 2) was prepared fresh from reagent grade









salts and deionized water. All chemicals were obtained from

Sigma Chemical Co. (St. Louis, MO, 63178). The pH of the

nutrient solution was maintained at 6.0 for the temperature,

humidity, and HID lamp experiments, but adjusted to the

appropriate [H+] during the experiments on the effects of

the [H+] on disease. The [H+] was adjusted in all

experiments using 0.01 N HNO3 and 0.02 N KOH.

Inoculum of E. solani f. sp. phaseoli (isolate F-28A,

obtained from A. J. Anderson, Utah State University, Logan,

Utah) was prepared on a modified carnation leaf agar (MCLA)

(Chapter 2) and consisted exclusively of macroconidia.

Inoculum in the stock suspension in deionized water was

adjusted to a density of 5.6 x 104 propagules/ml. A

hemacytometer was used to count the numbers of propagules/ml

of solution in this and all subsequent experiments. The

inoculum density of the stock suspension was confirmed by

serial dilutions and a micro-drop counting procedure in

which 10, 1-pl samples from the inoculum stock suspension

were placed on the surface of nutrient solution agar (NSA)

(Chapter 2); the number of macroconidia in each microdrop

was then counted. Plants were inoculated in 50% of the

experimental units per HID lamp-zone or growth chamber by

injecting 50 ml of inoculum as five, 10-ml samples into each

plastic container.

To determine the fluctuations in the numbers of

propagules of E. solani f. sp. phaseoli in the nutrient

solution of each treatment, three, 1-ml samples were taken










from each of two experimental units per growth chamber or

HID lamp-zone at 0, 1, 5, 10, and 14 days after inoculation.

Each sample was dispensed onto five plates of potato

dextrose agar supplemented with 50.0 mg of chlortetracycline

hydrochloride and 1.0 ml of tergitol NP-10 per liter of

medium (PDATC) and incubated under low light (< 10 Amol*
s-1.m-2) for 5-7 days. Colonies of E. solani f. sp.

phaseoli were counted for each sample date; isolates of

other fungal species were obtained from these cultures and

identified to genus using available keys

(12,36,42,61,160,229).

Plants were incubated for 14 days under various

combinations of temperature, [H+], humidity, and light

quality, as will be described below. The numbers of plants

exhibiting wilt symptoms at 14 days were counted for each

treatment. Shoots and roots were measured individually for

fresh and dry weights. Data were pooled and adjusted to

yield mean plant fresh and dry weights per each experimental

unit. Root samples were obtained from two plants per

experimental unit to visually determine the presence of

macroconidia and conidiophores produced on root surfaces.

Root samples were taken between 7 and 14 days after

inoculation from inoculated plants maintained at 20 C and

from all treatments at 14 days. Root samples were stained

in 0.05% trypan blue (Chapter 2) and viewed at various

magnifications with a Nikon Optiphot compound microscope.

The morphometric method of root analysis described by Fitter










(70) was utilized throughout these experiments. To deter-

mine the percentage of roots colonized by E. solani f. sp.

phaseoli, three, 1.0-cm-long root segments were excised from

symptomatic and asymptomatic roots from both noninoculated

and inoculated treatments. Root segments were placed on

PDATC and incubated under low light for 5-7 days.

Root segments were prepared for scanning electron

microscopy (SEM) by fixation in phosphate buffered 3%

glutaraldehyde and 1% osmium tetroxide (Chapter 5). Samples

were dehydrated in ethanol and critical-point-dried with

CO2. Specimens were mounted, coated with gold, and viewed

with a Hitachi S-530 scanning electron microscope, as

described in Chapter 5.

A bioassay was developed to determine if plant growth-

promoting or growth-inhibiting compounds, that could have

contributed to differences in plant weights among

treatments, had accumulated in the nutrient solution of

various treatments. The bioassay was modified from the

procedures of Bentley and Bickle (16). Two hundred

milliliters of nutrient solution were collected from each

experimental unit of both noninoculated and inoculated

treatments. Samples were bulked for each treatment and

filter-sterilized through 0.45-Mm, Gelman Mini-Capsule,

cartridge filters (Gelman Sciences, Inc., Ann Arbor, MI,

48106). Bulked samples were stored at 4 C until used in the

bioassays.










For each bioassay sample tested, 100 ml of nutrient

solution (pH 6.0) containing 3 g Bacto agar (Difco

Laboratories, Detroit, MI, 48232) were placed in a 500-ml

flask and autoclaved for 20 min at 1.1 kg*cm-2 and 240 C.

The agar was allowed to cool to 60 C, and 100 ml of test

nutrient solution from an individual treatment were added to

the flask; agar was poured immediately into two, square

polystyrene petri dishes. Prior to the addition of the

bioassay sample to the autoclaved agar, it was adjusted to

pH 6.0 and sterilized a second time using a 0.45-Am,

cellulose nitrate filter and Nalgene* PSF filtration system

(Baxter Healthcare Corp., McGraw Park, IL, 60085). A

separate sample of NSA was prepared from 200 ml of fresh

nutrient solution to serve as a nonexperimental control in

the event that both inoculated and noninoculated treatments

were similar in their effects on germinating seed.

Seeds of Lactuca sativa L., cv. 'Great Lakes,' were

surface-sterilized for 15 min with intermittent agitation in

a solution of 0.13% NaOC1 (2.5% bleach, Kare Chemical Co.,

Opalocka, FL, 33054) with Tween-20 (J.T. Baker Chemical Co.,

Phillipsburg, NJ, 08865) added at the rate of 1.0 ml*L-1.

Seeds were rinsed three times in sterile deionized water and

allowed to imbibe water for 6-8 hr. Twenty-five seeds were

placed on each of two plates of bioassay media prepared from

each treatment. In preliminary studies, the lettuce

cultivar Great Lakes was selected based on its sensitivity










to 2,4,5-triphenyl-2H-tetrazolium chloride at a concentra-

tion of 0.05 mg*L-1 (data not shown).

Plates were wrapped with parafilm and incubated at 25 C

in a Percival I-30BLL incubator (Percival Manufacturing Co.,

Boone, IA 50036) for 4 days for the temperature experiments

and 3 days for the [H+], humidity, and light quality

experiments. A 12-hr, diurnal light cycle was provided

using four, 20-watt, fluorescent bulbs, which yielded a

photon-flux of 50 pmol*s-1*m-2. All bioassay plates were

inclined 45 during incubation. After incubation, root tips

were marked and the lengths of the seedling radicles were

measured using a Sony DXC-3000 color video camera (Sony

Corporation, Japan) electronically coupled to the Microcompe

Integrated Video Image Analysis System (Southern Micro

Instruments, Inc., Atlanta, GA, 30339), as was described

previously (Chapter 2).

Statistical analyses were conducted with the

Statistical Analysis System (SAS) (SAS Institute, Inc.,

Cary, NC, 27512). Unless otherwise stated, statistical

significance was at the P 0.05 level. Square-root or log

transformations were utilized in most experiments to induce

homogeneity of variances among treatments. A 0.25-power

transformation was appropriate for a few experiments because

square-root and log transformations, respectively, either

under- or over-transformed the data. Plots of residuals and

predicted values were used to determine the appropriate

transformation for each experiment (213). Orthogonal










polynomial contrast analyses were used when treatments were

equally spaced and quantitatively related (213); standard

Analysis of Variance was used for data that were not

quantitatively related. Least-squares mean separation tests

were employed where significant F-values indicated treatment

effects.

A split-plot design was used for the study on the

effects of temperature on disease; temperature (20, 24, 28,

32, and 36 C) was the main plot and inoculum level (0 and

500 propagules/ml) the split plot. Temperature was

replicated three times temporally, while inoculum level was

replicated four times within each repitition of the main

plot. The experimental mean for humidity, across all

temperatures tested, was 12.0 mm Hg VPD (50% relative

humidity at 25 C).

A split-split-plot design was used for the studies on

the effects of [H+], humidity, and light quality on disease.

In the experiments on [H+], temperature (20 and 25 C) was

the main plot, [H+] (pH 4.0, 5.0, 6.0, and 7.0) was the

split plot, and inoculum level (0 and 500 propagules/ml) was

the split-split plot. Temperature, inoculum level, and [H+]

each were replicated twice, and each test within the

experiment was conducted three times. During these tests

humidity was maintained at a VPD of 6.0 mm Hg.

In the experiments on humidity, temperature (20, 25,

and 30 C) was the main plot, humidity (6.0 and 12.0 mm Hg,

VPD) was the split plot, and inoculum level (0 and 500










propagules/ml) was the split-split plot. Temperature,

humidity, and inoculum level each were replicated two times

temporally.

In experiments on the effects of light quality on

disease, temperature (20 and 25 C) was the main plot, HID

lamp type was the split plot, and inoculum level (0 and 500

propagules/ml) was the split-split plot. Temperature and

light quality each were replicated three times temporally,

while inoculum level was replicated four times within each

repitition of the main plot. During these tests humidity

was maintained at a VPD of 6.0 mm Hg.

Trifoliate leaves of Y. radiata from experiments on the

effects of temperature, [H+], humidity, and light quality

were oven-dried at 70 C for 3-5 days. Replicate samples

from each treatment were bulked and then ground to pass a

40-mesh metal screen using a Whiley mill. Elemental

analysis was conducted by Dr. Wade Berry, University of

California, Berkeley, CA, using an optical emission

spectrometry system (3). Ion composition of trifoliate

leaves from experiments on the effects of temperature,

humidity, and [H+] on disease were analyzed using orthogonal

polynomial contrasts; data from the experiment on the effect

of light quality on disease were analyzed using Analysis of

Variance.










Results

Shoot weights, root weights, whole plant weights, and

shoot-root ratios were analyzed separately for each

experiment. Most treatment effects were consistent,

regardless of plant part analyzed; whole plant fresh weights

are presented for simplicity. Whole plant dry weights and

shoot-root ratios are presented in Appendix A.

Growth of noninoculated V. radiata plants was influenced by

temperature, but not by [H+], humidity, and light quality

(Tables 3-1, 3-2, 3-3, and 3-4). Optimum growth occurred

between 28-32 C (Table 3-1). Noninoculated plants grown at

20 C were approximately 30-35% as large as plants grown at

28-32 C (Tables 3-1 and 3-3).

The severity of disease caused by E. solani f. sp.

phaseoli in Y. radiata was influenced by temperature (Table

3-1) and [H+] (Table 3-2), but not by humidity (Table 3-3)

and light quality (Table 3-4). Disease was greatest at 20 C

for all experiments (Table 3-1, 3-2, 3-3, and 3-4); between

52 and 97% of inoculated plants wilted by 14 days after

inoculation, and plant fresh weights were reduced an average

of 60-75% compared to noninoculated plants at 20 C.

Differences between noninoculated and inoculated plants at

24-28 C also were significant (P 0.05). Weights of

noninoculated and inoculated plants at 30 C, or above, were

not different (P > 0.10) (Table 3-1 and 3-3). The effects

of temperature between 20 and 36 C (Table 3-1) were best

described by quadratic polynomial equations for









Table 3-1. Effects of temperature on disease caused by
Fusarium solani f. sp. phaseoli in Viana radiata.

Temperature Plant fresh weight (g)
(C)
Noninoculated Inoculated

20 4.45 az 1.76* a

24 8.58 b 6.73* b

28 13.44 d 11.11* d

32 13.71 d 13.40 e

36 10.48 c 10.84 c

Z Table values represent the means of 12 replicates per
treatment (four replicates per treatment in each of three
experiments). Data were transformed to logs and then
subjected to an orthogonal polynomial contrast analysis;
table values are presented as detransformed numbers.
Least-squares mean separation tests were used to examine
pairwise comparisons between appropriate treatments.
Significant differences in the comparisons between
noninoculated and inoculated plants at each temperature
are indicated by an asterisk (*) (P : 0.05). Treatments
in columns followed by the same letter were not different
(P > 0.05).









Table 3-2. Effects of hydrogen ion concentration and
temperature on disease caused by Fusarium solani f. sp.
Dhaseoli in Vigna radiata.

pH Temperature Plant fresh weight (g)
(C)
Noninoculated Inoculated

4.0 20 4.23 aZ 1.52* a

25 9.99 b 9.74 c

5.0 20 4.36 a 1.08* a

25 10.90 b 8.28* bc

6.0 20 4.61 a 1.25* a

25 10.49 b 8.49* bc

7.0 20 4.44 a 1.07* a

25 9.78 b 7.13* b

z Table values represent the means of six replicates per
treatment (two replicates per treatment in each of three
experiments). Data were adjusted with a 0.25-power
transformation and then subjected to an orthogonal
polynomial contrast analysis; table values are presented
as detransformed numbers. Least-squares mean separation
tests were used to examine pairwise comparisons between
appropriate treatments. Significant differences in the
comparisons between noninoculated and inoculated plants
for each temperature and hydrogen ion concentration are
indicated by an asterisk (*) (P < 0.05). Treatments in
columns followed by the same letter were not different
(P > 0.05).









Table 3-3. Effects of humidity and temperature on disease
caused by Fusarium solani f. sp. phaseoli in Vinna radiata.

Humidity Temperature Plant fresh weight (g)
(mm Hg, VPD)Y (C)
Noninoculated Inoculated

6.0 20 4.98 az 1.62* a

25 13.44 b 9.61* b

30 15.18 b 15.77 c

12.0 20 4.78 a 1.72* a

25 13.62 b 9.76* b

30 14.92 b 14.96 c

y Humidity was measured as vapor pressure deficit (VPD) in
mm Hg; vapor pressure deficits of 6.0 and 12.0 mm Hg, at
25 C, correspond to 75% and 45% relative humidities,
respectively.
z Table values represent the means of eight replicates per
treatment (four replicates per treatment in each of two
experiments). Data were transformed to logs and then
subjected to an orthogonal polynomial contrast analysis;
table values are presented as detransformed numbers.
Least-squares mean separation tests were used to examine
pairwise comparisons between appropriate treatments.
Significant differences in the comparisons between
noninoculated and inoculated plants at each temperature
and humidity are indicated by an asterisk (*) (P 5 0.05).
Treatments in columns followed by the same letter were
different (P > 0.05).









Table 3-4. Effects of light quality and temperature on
disease caused by Fusarium solani f. sp. phaseoli in Viana
radiata.

Lamp-type Temperature Plant fresh weight (g)
(C)
Noninoculated Inoculated

Fluorescent 20 4.76 az 1.60* a

25 9.49 b 5.77* b

Metal halide 20 4.51 a 1.57* a

25 8.93 b 5.75* b

High pressure 20 5.09 a 1.71* a
sodium
25 11.16 b 7.53* b

z Table values represent the means of 12 replicates per
treatment (four replicates per treatment in each of three
experiments). Data were transformed to logs and then
subjected to an Analysis of Variance and a least-squares
mean separation test; table values are presented as
detransformed numbers. Significant differences in the
comparisons between noninoculated and inoculated
treatments at each temperature and lamp-type are indicated
by an asterisk (*) (P < 0.05). Treatments in columns
followed by the same letter were not different (P > 0.05).










noninoculated and inoculated plants (P < 0.001). Plant wilt

varied among experiments and was not considered a reliable

estimate of the severity of disease because at 25 C plants

periodically would recover from wilt by initiating lateral

roots from their lower stems just above symptomatic tissues.

Necrotic flecks were observed at 20 and 25 C on all

root orders 72-96 hr after inoculation. At 20 C, necrotic

flecks coalesced into larger necrotic lesions by 5 days, and

first- and second-order roots were moribund by 10 days after

inoculation. At 24 and 28 C, more severe symptoms developed

on second-order roots with root hairs than developed on

first- or second-order roots without root hairs. Lower

hypocotyls were less affected by E. solani f. sp. Dhaseoli

than the root systems; tissue necrosis usually did not

develop acropetally to plant crowns. Root symptoms were

most severe at 20 C and declined dramatically as temperature

increased; no root symptoms were observed on lateral roots

at 30 C or above. Small necrotic lesions were observed on

the main tap roots just below the crown at 30 C, but not at

32 and 36 C. When root necrosis occurred, it was most

severe in the crown area of plants, regardless of the

treatment parameter. At 24 and 28 C, plants initiated new

lateral roots from severely damaged tissues, presumably from

intact pericycle cells, between 10 and 14 days after

inoculation.

Conidiophores and macroconidia were produced between 10

and 14 days after inoculation at 20 C and by 14 days at 24










and 28 C. Conidiophores and macroconidia were most abundant

on heavily damaged roots maintained at 20 C; they were less

abundant as temperature increased to 28 C, and they were

absent from inoculated roots at 30-36 C. Highly branched

monophialides and sporodochia (Figure 3-1) were observed

between 20 and 25 C on second-order roots, but not at 28 C

and not on any other root orders. Branched monophialides

emerged directly from the root epidermis, root tips, and

from cortical cavities formed when longitudinal splits

occurred in the root epidermis on severely damaged roots

(Figure 3-1). At 20-28 C unbranched monophialides were

produced from individual hyphae on the root surface, termed

rhizosphere hyphae to differentiate them from hyphae

emanating from cortical cavities (Figure 3-1C).

Microconidia were not observed in the rhizosphere under any

environmental conditions; chlamydospores were only rarely

observed at 20 C. Development of branched and unbranched

monophialides, sporodochia, and secondary macroconidia was

consistent at each temperature, but was unaffected by light

quality and humidity. Mature conidiophores and macroconidia

of F. solani f. sp. phaseoli were not observed at pH 4.0;

sporodochia and branched monophialides were disorganized and

poorly developed (Figure 3-2). Mature conidiophores and

macroconidia were formed at pH 5.0, 6.0. and 7.0, at both 20

and 25 C.

At temperatures between 20 and 28 C, Fusarium solani f.

sp. phaseoli colonized between 90 and 100% of symptomatic
































Figure 3-1. Conidiophores of Fusarium solani f. sp.
phaseoli observed on severely damaged roots of Vigna
radiata. Mature macroconidia (ma) were produced from
conidiophores on roots grown at 20-28 C, but not on roots
grown at 30-36 C. A,B, Branched monophialides (bm) were
observed emerging from the root epidermis (ep), from root
tips (rt), and from cortical cavities (cc) formed on
severely damaged roots when longitudinal splits developed in
the rot epidermis. The lower left-hand corner of Figure
3-1A indicates a cortical cavity on the root of V. radiata.
C, Unbranched monophialides (um) developed from rhizospere
hyphae (rh). D, Sporodochia (sp) developed from hyphae
emerging from the root epidermis.





63


i LI
































Figure 3-2. Deformed conidiophores of Fusarium solani f.
sp. phaseoli on roots of Viqna radiata grown at 20 C and pH
4.0. A, Conidiophores (co) formed branched complexes (bc)
similar to branched monophialides at other hydrogen ion
concentrations, but mature macroconidia were not produced.
B, Deformed conidiophores were observed within cortical
cavities (cc) formed on severely damaged roots when
longitudinal splits developed in the root epidermis (ep). C,
Mycelia also remained disorganized in cortical cavities and
did not develop rudimentary conidiophores.







65
















im












W7








NOW.










roots and between 21 and 83% of asymptomatic roots (Tables

3-5, 3-6, 3-7, and 3-8). The fungus was not isolated from

asymptomatic roots at 36 C, but it was isolated from

asymptomatic roots at all other temperatures. The decrease

in the percentage of asymptomatic roots colonized by E.

solani f. sp. phaseoli at 24-25 C as compared to 20 C was

generally significant for three experiments (P S 0.05)

(Tables 3-5, 3-7, and 3-8), but was not significant for the

experiment on the effects of [H+] on disease (Table 3-6).

An effect of [H+] on disease was observed at 25 C, but

not at 20 C (Table 3-2). At 20 C, disease was similar among

all [H+] tested and was consistent with other experiments in

which treatments were maintained at 20 C. At 25 C, fresh

weights of noninoculated and inoculated plants were not

different at pH 4.0 (P > 0.10), but they were different at

pH 5.0, 6.0, and 7.0 (P < 0.05). Fresh weights of

inoculated plants at 25 C and pH 4.0 were larger than fresh

weights of inoculated plants at 25 C and pH 7.0. Root

symptoms were dramatically reduced on inoculated plants

incubated at 25 C and pH 4.0, compared to root symptoms on

plants incubated at 25 C and pH 5.0, 6.0, and 7.0. The

individual effects of temperature and [H+] were best fit by

linear polynomial equations (P s 0.05). An interactive

effect between temperature and [H+] was not observed

(P > 0.10).

Numbers of propagules (colony-forming units/ml) of the

pathogen detected in nutrient solution at time zero









Table 3-5. Effects of temperature on the colonization of
roots of Vigna radiata by Fusarium solani f. sp. phaseoli.

Temperature Percent of colonized root segments
(C) Symptomatic Asymptomatic
roots roots

20 100 ay 59 b

24 100 a 21 c

28 94 a 49 b

32 _-z 38 bc

36 -- Od

y Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of F. solani f. sp.
phaseoli. Table values represent the means of three
replicates per treatment (one replicate per treatment for
each of three experiments). Data were transformed to the
arcsines of square roots; table values are presented as
detransformed numbers. Data from asymptomatic roots were
subjected to an orthogonal polynomial contrast analysis
and least-squares mean separation tests. Comparisons
between treatments equal to 100% and treatments < 100%
were conducted using paired t-tests for comparing
treatment means to a standard value; significant test
statistics were based on the standard errors of the means
of the normally distributed populations. Treatments
followed by the same letter were not different (P > 0.05).
z Symptomatic roots were not observed at 32 and 36 C.









Table 3-6. Effects of hydrogen ion concentration and
temperature on the colonization of roots of Vigna radiata by
Fusarium solani f. sp. phaseoli.

pH Temperature Percent of colonized root segments
(C)
Symptomatic Asymptomatic
roots roots

4.0 20 92 aZ 63 b

25 100 a 75 b

5.0 20 100 a 46 b

25 100 a 36 b

6.0 20 98 a 63 b

25 100 a 47 b

7.0 20 100 a 72 b

25 100 a 44 b

z Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of F. solani f. sp.
phaseoli. Table values represent the means of three
replicates per treatment (one replicate per treatment for
each of three experiments). Data were transformed to the
arcsines of square roots; table values are presented as
detransformed numbers. Data from asymptomatic roots were
subjected to an orthogonal polynomial contrast analysis
and least-squares mean separation tests. Comparisons
between treatments equal to 100% and treatments < 100%
were conducted using paired t-tests for comparing
treatment means to a standard value; significant test
statistics were based on the standard errors of the means
of the normally distributed populations. Treatments
followed by the same letter were not different (P > 0.05).









Table 3-7. Effects of humidity and temperature on the
colonization of roots of Vigna radiata by Fusariu solani f.
sp. phaseoli.

Humidity Temperature Percent of colonized root
segments
(mm Hg, VPD)x (C) Symptomatic Asymptomatic
roots roots

6.0 20 98 ay 60 bc

25 90 a 35 d

30 __z 60 bc

12.0 20 94 a 60 bc

25 90 a 46 cd

30 -- 70 b

x Humidity was measured as vapor pressure deficit (VPD) in
mm Hg; vapor pressure deficits of 6.0 and 12.0 mm Hg, at
25 C, correspond to 75 and 45% relative humidities,
respectively.
Y Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of E. solani f. sp.
Dhaseoli. Table values represent the means of two
replicates per treatment (one replicate per treatment for
each of two experiments). Data were transformed to the
arcsines of square roots and then subjected to an
orthogonal polynomial contrast analysis and least-squares
mean separation tests; table values are presented as
detransformed numbers. Treatments followed by the same
letter were not different (P > 0.05).
z Symptomatic roots were not observed at 30 C.









Table 3-8. Effects of light quality and temperature on the
colonization of roots of Vina radiata by Fusarium solani f.
sp. phaseoli.

Lamp-type Temperature Percent of colonized root
segments
(C) Symptomatic Asymptomatic
roots roots

Fluorescent 20 96 az 65 ab

25 95 a 42 b

Metal halide 20 96 a 84 a

25 97 a 32 c

High pressure 20 93 a 66 a
sodium
25 97 a 36 c

z Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of E. solani f. sp.
phaseoli. Table values represent the means of three
replicates per treatment (one replicate per treatment for
each of three experiments). Data were transformed to the
arcsines of square roots and then subjected to an Analysis
of Variance procedure; table values are presented as
detransformed numbers. Treatments followed by the same
letter were not different based on least-squares mean
separation tests (P > 0.05).









increased as temperature increased during experiments on

temperature, [H+], and humidity (P s 0.05) (Tables 3-9, 3-

10, and 3-11), but not light quality (Table 3-12). The

relationships between temperature and the numbers of

propagules detected were best described by linear polynomial

equations (P < 0.03) (Tables 3-9 and 3-11). After 24 hr,

the numbers of propagules detected in nutrient solution

decreased dramatically in all experiments and remained low

for 1-10 days after inoculation in most experiments (Tables

3-9, 3-10, and 3-12). Numbers of propagules detected in

nutrient solution generally increased between 10 and 14 days

at 20 C (Tables 3-9, 3-10, and 3-12), except when the [H+]

was maintained at pH 4.0 and in the experiments with

humidity (Tables 3-10 and 3-11).

Species of Acremonium, Aspercillus, Cephalosporium,

Chaetomium, Cladosporium, Curvularia, Cylindrocarpon,

Fusarium, Mucor, Penicillium, Stachybotrys, and Trichoderma

were isolated concommittantly with F. solani f. sp. phaseoli

from both inoculated and noninoculated treatments. Fusarium

spp., other than f. solani f. sp. phaseoli, isolated from

nutrient solution were identified to species based on their

growth on carnation leaf agar using the keys of Nelson

et al. (160); all isolates were keyed to Fusarium oxysporum.

Species of Aspergillus, Chaetomium, Cladosporium, and

Penicillium were isolated most frequently; the numbers of

propagules detected in nutrient solution were generally

between 5 and 50 colony-forming units (cfu)/ml.











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Periodically the numbers of propagules of CladosDorium spp.

detected in nutrient solution exceeded 200 cfu/ml. Fungal

contaminants were not correlated with any specific

treatment, nor were fungi correlated to anomalous root

symptoms on noninoculated plants. Carbon-based materials in

contact with the nutrient solution were found to be the

primary source of fungal contamination. Plastic tubing,

foam insulation, and black conduit tape were readily

colonized by fungi. Plastic tubing was periodically

replaced to reduce fungal contamination in the nutrient

solution. Species of Cladosporium, Chaetomium, and

Trichoderma were isolated most frequently from plastic

tubing and foam insulation; Cladosporium spp. were the

predominant fungi isolated from black conduit tape placed on

the upper surface of the 4-L tanks to reduce light

penetration into the hydroponic solutions.

Root length of L. sativa, cv 'Grand Rapids,' was not

significantly affected in bioassays by temperature, [H+1],

humidity, and light quality experiments. Bioassays were

generally consistent between treatments within each

experiment, but differed between experiments. In the

experiments on the effects of temperature on disease, root

length of L. sativa varied between 23.4 and 27.3 mm after 4

days of incubation. In other experiments bioassays were

incubated for 3 days and root length was shorter, but root

length did not differ between treatments within each

experiment. Root length varied between 18.7 and 21.3 mm for










the [H+] and light quality experiments, and between 12.9 and

17.0 mm for the humidity experiment.

Temperature generally imparted the greatest affect on

element composition in foliar tissues of noninoculated

plants of Y. radiata (Tables 3-13, 3-14, 3-15, and 3-16).

Boron and iron were positively correlated to increasing

temperature and were best described by linear polynomial

equations (P < 0.01) (Tables 3-13 and 3-15). Molybdenum was

negatively correlated to increasing temperature and was best

described by a linear polynomial equation (P < 0.01).

Calcium, potassium, and silicon were best described by

positive quadratic polynomial equations (P < 0.01), with

maximum concentrations observed between 24 and 32 C.

Copper, magnesium, and zinc were best described by negative

quadratic polynomial equations (P < 0.03), with minimum

concentrations observed between 24 and 32 C. The most

complex response to temperature was observed for Mn, which

increased in concentration between 20 and 24 C, decreased

between 24 and 28 C, and then increased between 28 and 36 C;

the response was best described by a cubic polynomial

equation (P < 0.01). No effect of temperature was observed

for either Na or P in noninoculated plants (P > 0.10).

Differences in element concentration between

noninoculated and inoculated plants were greatest at 20 C

for Ca, Cu, Fe, K, Mn, Na, P, Si, and Zn (P 5 0.05) (Tables

3-13, 3-14, 3-15, and 3-16). Differences in element













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concentration between noninoculated and inoculated plants

were not observed for B and Mg for any temperature

(P > 0.10), except for B at 20 C and pH 7.0 (Table 3-14).

Differences in element concentration between noninoculated

and inoculated plants generally decreased as temperature

increased in all experiments (Tables 3-13, 3-14, 3-15, and

3-16); element concentration of tissues from noninoculated

and inoculated plants were similar at 28-36 C (P > 0.10)

(Table 3-13).

Effects of [H+] on element concentration were not

observed for Ca, Mo, P, Si, Na, and Mg (P > 0.05) (Table

3-14). Boron was positively correlated with increasing pH

and its response was best described by a linear polynomial

equation (P = 0.019). Copper, iron, potassium, manganese,

and zinc decreased with increasing pH at both 20 and 25 C

and the responses were best described by linear polynomial

equations (P < 0.05). Differences in element concentration

between noninoculated and inoculated plants were greatest at

20 C and appeared to be little affected by [H+]

(Table 3-14).

Effects of humidity on element concentration in either

noninoculated or inoculated plants were not observed for any

elements tested. Differences between noninoculated and

inoculated plants were generally not affected by humidity

(Table 3-15) nor by light quality (Table 3-16). Effects of

light quality on element concentration were not observed for

Ca, Cu, K, and Na (P > 0.10) (Table 3-16). Iron and silicon










were suppressed, and Mn, P, and Zn increased, in plants

grown under MH lamps (P < 0.05). Boron and magnesium were

slightly lower in plants grown under HPS lamps compared to

FL and MH lamps (P < 0.05). The accumulation of Mo was

slightly increased in plants grown under FL lamps compared

to MH and FL lamps (P < 0.05).


Discussion

Microbial contamination of space-based CELSS will

likely include fungi that are ecologically adapted to

survival on plants originally grown in terrestrial

environments. For example, 29 of 57 genera of fungi

isolated during the Apollo 14 and 15 missions (218) were

found in the United States Department of Agriculture (USDA)

host index of plant diseases (150). It is difficult to

ascertain if microbial contamination of CELSS modules will

constitute a realistic threat to plant health. Microgravity

may alter many aspects of plant-microbe interactions such

that new and unique pathological relationships develop.

Although, the physiologies of plants (46,87,121,155,156,202)

and microbes (2,170,214,216,240) are altered by

microgravity, no studies on plant-microbe interactions in

the space environment have been reported.

As stated earlier, hydroponic plant production systems

will be utilized in space-based CELSS (30,101,124,166,219),

and it seems apparent that a clear understanding of disease

etiology, epidemiology, and control in terrestrial




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81,9(56,7< 2) )/25,'$


EFFECTS OF TEMPERATURE AND HYDROGEN ION CONCENTRATION ON
DISEASE CAUSED BY FUSARIUM SOLANI F. SP. PHASEOLI IN VIGNA
RADIATA GROWN IN HYDROPONIC NUTRIENT SOLUTION
v
by
ANDREW CONRAD SCHUERGER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991

ACKNOWLEDGMENT S
The author would like to express his sincere gratitude
to Dr. David J. Mitchell for his constant patience, honesty,
and moral support during the course of this project. This
research certainly would have been more difficult if it were
not for the friendship that Dr. Mitchell extended to that
young student with the unusual ideas so many years ago. The
author also would like to thank the other members of the
supervisory committee, Drs. R. Charudattan, James 0.
Strandberg, and David M. Sylvia, for their patience and
suggestions. Special thanks are due Dr. David T. Kaplan for
his suggestions in the experiments on spore attachment to
plant roots.
Appreciation is extended to the Walt Disney World, Co.,
which has provided financial support for this research, and
to the Biological Science Office of the National Aeronautics
and Space Administration (NASA) at the Kennedy Space Center,
FL, which has provided in-kind support under a joint
agreement between the Walt Disney World, Co., and NASA. In
addition, thanks are due Dr. Henry A. Robitaille, Manager of
Science and Technology, EPCOT Center, and Dr. William M.
Knott, III, Biological Science Officer, NASA, Kennedy Space
Center, for their suggestions and support for this research.
Special thanks are given to Dr. Frederick L. Petitt,
Manager of The Land, EPCOT Center, and Steven Linda,
ii

Deptartment of Statistics, University of Florida, for their
suggestions and assistance in the analysis of experimental
data. Their assistance greatly improved the quality of this
research. Furthermore, appreciation is extended to Dinah
Jordan, for her assistance in the preparation of this
manuscript, and to the plant pathology staff at The Land,
Jean Carlson-Batzer, William Hammer, Kristin Pategas, and
Bret Norman, who performed their jobs with the excellence
that created the freedom to pursue this project. Thanks
also are due the plant pathology students, Karen Wendt,
Andrew Deckard, Bryan Kleiwer, Phil Liable, Andrew Hoetger,
and James Adams, who helped in various ways.
The author also would like to acknowledge the
contributions of scientists and philosophers, both living
and deceased, who have provided the intellectual and
metaphysical framework of human knowledge. We seldom
recognize that our progress would be nearly futile if it
were not for the passions and tribulations of those who have
come before us.
iii

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES ix
ABSTRACT xi
CHAPTERS
1 INTRODUCTION 1
2 EFFECTS OF CULTURE MEDIA ON MACROCONIDIUM
MORPHOLOGY AND PATHOGENICITY OF FUSARIUM SOLANI
F. SP. PHASEOLI 11
Introduction 11
Materials and Methods 12
Results 30
Discussion 38
3EFFECTS OF TEMPERATURE, HYDROGEN ION
CONCENTRATION, HUMIDITY, AND LIGHT QUALITY ON
DISEASE CAUSED BY FUSARIUM SOLANI F. SP.
PHASEOLI IN VIGNA RADIATA 42
Introduction 42
Materials and Methods 45
Results 54
Discussion 87
4EFFECTS OF TEMPERATURE AND HYDROGEN ION
CONCENTRATION ON ATTACHMENT OF MACROCONIDIA OF
FUSARIUM SOLANI F. SP. PHASEOLI TO ROOTS OF VIGNA
RADIATA IN HYDROPONIC NUTRIENT SOLUTION 102
Introduction 102
Materials and Methods 104
Results 115
Discussion 128
IV

5THE INFLUENCE OF SECRETED SPORE MUCILAGE OF
MACROCONIDIA OF FUSARIUM SOLANI F. SP.PHASEOLI
ON SPORE ATTACHMENT TO ROOTS OF VIGNA
RADIATA IN HYDROPONIC NUTRIENT SOLUTION 141
Introduction 141
Materials and Methods 143
Results 148
Discussion 166
6 EFFECTS OF CARBON SOURCE ON SPORE ATTACHMENT AND
GERMINATION OF MACROCONIDIA OF FUSARIUM SOLANI
F. SP. PHASEOLI ON ROOTS OF VIGNA RADIATA IN
HYDROPONIC NUTRIENT SOLUTION 173
Introduction 173
Materials and Methods 176
Results 184
Discussion 193
7 SUMMARY AND CONCLUSIONS 199
APPENDICES
A DRY WEIGHTS AND SHOOT-ROOT RATIOS FROM
PATHOGENICITY TESTS 208
B DRY WEIGHTS AND SHOOT-ROOT RATIOS FROM
SPORE ATTACHMENT EXPERIMENTS 217
LITERATURE CITED 221
BIOGRAPHICAL SKETCH 243
v

LIST OF TABLES
Table Page
2-1. Production of macroconidia, microconidia, and
chlamydospores by 43 strains of Fusarium spp.
grown for 14 days on modified carnation leaf
agar (MCLA), carnation leaf agar (CLA), and
potato dextrose agar (PDA) 15
2-2. Fusarium spp. tested for pathogenicity on
various hosts in a hydroponic system 20
2-3. Length and width of macroconidia of Fusarium
solani f. sp. phaseoli grown on four
different media 36
2-4. Effects of culture media on the virulence of
inoculum of Fusarium solani f. sp. phaseoli and
on disease in Viana radiata 37
3-1. Effects of temperature on disease caused by
Fusarium solani f. sp. phaseoli in
Viqna radiata 56
3-2. Effects of hydrogen ion concentration and
temperature on disease caused by Fusarium
solani f. sp. phaseoli in Viqna radiata 57
3-3. Effects of humidity and temperature on disease
caused by Fusarium solani f. sp. phaseoli in
Viqna radiata 58
3-4. Effects of light quality and temperature on
disease caused by Fusarium solani f. sp.
phaseoli in Viqna radiata 59
3-5. Effects of temperature on the colonization
of roots of Viqna radiata by Fusarium solani
f. sp. phaseoli 67
3-6. Effects of hydrogen ion concentration and
temperature on the colonization of roots of
Viqna radiata by Fusarium solani f. sp. phaseoli 68
3-7. Effects of humidity and temperature on the
colonization of roots of Viqna radiata by
Fusarium solani f. sp. phaseoli 69
vi

3-8. Effects of light quality and temperature on
the colonization of roots of Viqna radiata by
Fusarium solani f. sp. phaseoli 70
3-9. Effects of temperature on the numbers of propa-
gules of Fusarium solani f. sp. phaseoli detected
in nutrient solution over time during experiments
on disease development in Viqna radiata 72
3-10. Effects of hydrogen ion concentration and
temperature on the numbers of propagules of
Fusarium solani f. sp. phaseoli detected in
nutrient solution over time during experiments on
disease development in Viqna radiata 73
3-11. Effects of humidity and temperature on the numbers
of propagules of Fusarium solani f. sp. phaseoli
detected in nutrient solution over time during
experiments on disease development in
Viqna radiata 74
3-12. Effects of light quality and temperature on the
numbers of propagules of Fusarium solani f. sp.
phaseoli detected in nutrient solution over time
during experiments on disease development in
Viqna radiata 75
3-13. Effects of temperature on the elemental
composition of foliage of Viqna radiata in
experiments on disease development by Fusarium
solani f. sp. phaseoli 78
3-14. Effects of hydrogen ion concentration and
temperature on elemental composition of foliage
of Viqna radiata in experiments on disease
development by Fusarium solani f. sp. phaseoli.. 80
3-15. Effects of humidity and temperature on the
elemental composition of foliage of Viqna radiata
in experiments on disease development by
Fusarium solani f. sp. phaseoli 82
3-16. Effects of light quality and temperature on the
elemental composition of foliage of Viqna
radiata in experiments on disease development
by Fusarium solani f. sp. phaseoli 84
4-1. Attachment of macroconidia of Fusarium solani
f. sp. phaseoli to four different root orders
and to the lower hypocotyl of Viqna radiata 118
4-2. Effects of temperature and hydrogen ion
concentration on the attachment of macroconidia
vii

of Fusarium solani f. sp. phaseoli to
second-order roots of Vigna radiata 120
4-3. Effects of temperature and hydrogen ion
concentration on germtube elongation of Fusarium
solani f. sp. phaseoli after spore attachment to
second-order roots of Vigna radiata 122
4-4. Effects of temperature and inoculum density on
the percentages of wilted plants of Vigna
radiata infected by Fusarium solani
f. sp. phaseoli 125
4-5. Effects of hydrogen ion concentration at the
time of inoculation on disease caused by
Fusarium solani f. sp. phaseoli
in Vigna radiata 127
4-6. Effects of hydrogen ion concentration at the
time of inoculation on the numbers of propagules
of Fusarium solani f. sp. phaseoli detected in
nutrient solution over time in experiments on
disease development in Vigna radiata 128
6-1. Effects of carbon source on the agglutination
of macroconidia of Fusarium solani f. sp.
phaseoli in nutrient solution and
root homogenate 179
6-2. Effects of macroconidium age on the agglutin¬
ation of spores of Fusarium solani f. sp.
phaseoli in nutrient solution after incubation
of macroconidia in root homogenate 185
6-3. Effects of hapten sugars on spore agglutination
and attachment of macroconidia of Fusarium
solani f. sp. phaseoli to roots of
Vigna radiata 187
viii

LIST OF FIGURES
Figure Page
2-1. Comparison of macroconidia of Fusarium solani
f. sp. phaseoli mounted on nutrient solution
agar and in a lactic acid, water, and
glycerol solvent 24
2-2. Schematic drawing of a cross section through
a seed germination tray used for the
production of seedlings of Vigna radiata 27
2-3. Comparison of macroconidia of Fusarium solani
f. sp. phaseoli produced on four different
culture media 34
3-1. Conidiophores of Fusarium solani f. sp.
phaseoli observed on severely damaged roots
of Vigna radiata 63
3-2. Deformed conidiophores of Fusarium solani
f. sp. phaseoli on roots of Vigna radiata grown
at 20 C and pH 4.0 65
3-3. Spectral photon fluxes (/Limol • s-1 *m-2) for
high pressure sodium, metal halide, and
fluorescent lamps 97
4-1. In vitro effects of temperature on radial
growth and germtube length of Fusarium solani
f. sp. phaseoli 116
4-2. In vitro effects of hydrogen ion concentration
on radial growth and germtube length of Fusarium
solani f. sp. phaseoli 117
4-3. Effects of inoculum density of Fusarium solani
f. sp. phaseoli and temperature on the severity
of disease in Vigna radiata 124
5-1. Effects of culture age on cytology of macro¬
conidia of Fusarium solani f. sp. phaseoli 150
5-2. Effects of age on the fixation and preparation
of macroconidia of Fusarium solani f. sp.
phaseoli for scanning electron microscopy 153
ix

5-3. Agglutination of macroconidia of Fusarium solani
f. sp. phaseoli incubated in nutrient solution
and root homogenate 155
5-4. Attachment of macroconidia of Fusarium solani
f. sp. phaseoli to root surfaces of
Vicma radiata 158
5-5. Agglutination of macroconidia of Fusarium
solani f. sp. phaseoli in root homogenate
maintained at 25 C and at different hydrogen
ion concentrations 160
5-6. Attachment of refuse from cultures of modified
carnation leaf agar (MCLA) to macroconidia of
Fusarium solani f. sp. phaseoli when
macroconidia were incubated in root homogenate.. 163
5-7. Production of spore mucilage at the tips of
macroconidia of Fusarium solani f. sp. phaseoli
during germination of conidia in root homogenate 166
6-1. Macroconidia of Fusarium solani f. sp. phaseoli
observed in the solutions of hapten sugars and
root homogenate 190
6-2. Macroconidia of Fusarium solani f. sp. phaseoli
observed after 24 hr in 50-mM solutions of
hapten sugars 192
x

Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
requirements for the Degree of Doctor of Philosophy
EFFECTS OF TEMPERATURE AND HYDROGEN ION CONCENTRATION ON
DISEASE CAUSED BY FUSARIUM SOLANI F. SP. PHASEOLI IN VIGNA
RADIATA GROWN IN HYDROPONIC NUTRIENT SOLUTION
by
Andrew Conrad Schuerger
August, 1991
Chairman: Dr. David J. Mitchell
Major Department: Plant Pathology
Disease caused by Fusarium solani f. sp. phaseoli in
Viqna radiata was greatest at 20 C and decreased with
increasing temperature until plant weight differences
between noninoculated and inoculated plants were not
significant at 30-36 C. The numbers of propagules of the
pathogen recovered from nutrient solution at 20 C increased
significantly between 10 and 14 days after inoculation, but
not at higher temperatures. Disease development was not
affected by humidity or light quality. Results of
bioassays, based on root lengths of germinating seedlings of
Lactuca sativa. did not support the hypothesis that plant
growth-inhibiting or growth-promoting compounds accumulated
in nutrient solution.
The effects of [H+] on disease development varied with
the timing of the [H+] treatments. In one experiment,
plants were inoculated at pH 6.0, and after 24 hr the
xi

nutrient soultions in separate treatments were adjusted to
pH 4.0, 5.0, 6.0, or 7.0. In a second experiment, plants
were inoculated at pH 4.0, 5.0, 6.0, or 7.0, and after 24 hr
the nutrient solutions in all treatments were adjusted to
pH 6.0. When the [H+] was adjusted to different levels
after inoculation, disease was less at pH 4.0 than at pH
5.0, 6.0, and 7.0. When the [H+] was adjusted to different
levels during inoculation, disease was less at pH 7.0 than
at pH 4.0, 5.0, and 6.0. The effects of [H+] on the
severity of disease in the second experiment coincided with
the effects of [H+] on spore attachment to roots of V.
radiata; both disease and the numbers of spores attached to
roots were greatest at pH 4.0.
Spore attachment to root surfaces, agglutination of
macroconidia in a root homogenate, secretion of a spore
mucilage during germination, and accretion of refuse to
spore tips were all suppressed at pH 3.0 and 7.0, and at
35 C. Based on the these responses, it was proposed that an
inducer may be present in roots of V. radiata that may be
involved in spore attachment, agglutination, and
germination. It was hypothesized that the inducer might be
a plant lectin. However, hapten sugars of plant lectins
failed to stimulate spore agglutination in nutrient
solution, agglutination in root homogenate, or spore
attachment to roots. Thus, the results did not support the
xii

hypothesis that lectins were involved in the attachment of
macroconidia of F. solani f. sp. phaseoli to roots of V.
radiata.
xiii

CHAPTER 1
INTRODUCTION
Controlled ecological life support systems (CELSS) have
been proposed for space bases to regenerate oxygen, water,
and food from human and industrial wastes (18,133,166).
Biological processes will be combined with physical and
chemical technologies to provide life support systems that
will reduce requirements for the resupply of materials from
Earth. Higher plants are considered the primary candidates
for photosynthesis in CELSS (85,101,166,178,219). Wheat,
rice, white potatoes, sweet potatoes, soybeans, peanuts,
lettuce, and sugar beets have been proposed as the primary
crops for CELSS (219). Hoff et al. (101) suggested several
additional species, including herb and fruit species, that
would increase the diversity of the crew's diet. Algal
species also may provide a portion of the crew's diet, but
no more than 10% of the human diet can be supplied by algae-
derived food products without physiological problems (166).
The National Aeronautics and Space Administration's
CELSS Breadboard Project at the Kennedy Space Center, FL,
was established to determine the best methods for growing
plants in a multispecies semi-closed system (178). A 7.5-m
x 3.5-m vessel, called the Biomass Production Chamber, was
l

2
modified for plant production, and research was initiated in
1986 with wheat. The production of edible biomass, the
regeneration of air and water, and the development of
monitoring and control systems were among the primary
objectives of the project (178).
Various methods of plant production in CELSS have been
proposed (30,56,166,178,235), but the precise method to be
used in a CELSS will depend on the specific mission. For
example, a plant production system in a microgravity
environment may require more stringent controls for the
containment of nutrient solution than a plant production
system might require on the moon. Wright et al. (235)
proposed a membrane system for growing plants in
microgravity that was based on the capillary forces observed
between fluids and solids. Plant roots were grown adjacent
to one side of a microporous acrylic membrane, and a
nutrient solution was maintained as a recirculating fluid on
the other side of the membrane. The laminar-flow of the
fluid along the inside surface of the membrane prevented
leakage of the fluid into the root environment by inducing a
slight negative pressure at the fluid-membrane interface.
When plant roots contacted the membrane, capillary forces
between the roots and the nutrient solution would draw the
fluid through the membrane, subsequently wetting the plant
roots. Dreschel and Sager (56) developed a similar system
using porous ceramic tubes. In addition, aeroponic methods,
nutrient film technique (NFT), and substrated hydroponic

3
systems have been proposed for plant production in
microgravity (30,178,166). Lunar regolith and synthetically
manufactured zeolites have been proposed as solid substrates
for plant production at a moon base (151). However, liquid
hydroponic systems appear to have several advantages over
solid substrates (30,166).
Advantages of liquid hydroponic systems compared to
substrated hydroponic systems may include 1) an increase in
nutrient and oxygen availability to roots in a liquid
system, 2) a reduction in root stress due to lower physical
resistance in a liquid matrix, 3) greater dilution of root
secretions in the nutrient solution, and 4) the ease of
recovery of the root biomass to facilitate rapid nutrient
recycling in a CELSS (30). The greatest disadvantage of
substrated hydroponic systems is the cost of lifting the
substrate into orbit (166). In addition, handling the
substrate during harvest and planting activities may be
difficult in microgravity (166). Thus, hydroponic systems
have been proposed as the primary plant production method in
CELSS (30,101,124,166,219).
Plants in CELSS may be threatened by microorganisms
transported into space as contaminants on spacecraft
components, equipment, astronauts, and plant-propagative
materials. Extensive microbial contamination has been
reported in American spacecraft (24,131,180,181,215,
217,218,228). Species of Alternaría. Aspergillus. Candida.
Cephalosporium. Cladosporium. Fusarium, Mucor. Penicillium.

4
Phoma. Saccharomvces. and Trichoderma were the most
prevalent fungi, and species of Bacillus. Escherichia.
Klebsiella. Micrococcus. Pseudomonas. Staphylococcus.
Serratia. and Streptococcus were the most prevalent bacteria
recovered from these systems. Strict quarantine procedures
were not effective in preventing contamination during these
missions. For example, 57 genera of fungi and actinomycetes
were recovered from human and spacecraft surfaces during the
Apollo 14 and 15 moon missions (218); contamination occurred
despite 3 weeks of quarantine for the crew and spacecraft
prior to the missions (215,216).
Whether microbial contamination of CELSS modules will
constitute a realistic threat to plant health is difficult
to ascertain. New concepts in microbial ecology and plant
pathology will undoubtedly be developed for CELSS.
Microgravity may alter aspects of plant-microbe interactions
such that new and unique pathological relationships develop.
Microgravity has been shown to influence the development,
cytology, and physiology of plants (46,87,156,155,121,202).
For example, the lignin content of developing hypocotyls and
the cell density of root tips were reduced when mung bean
seed were germinated in microgravity (46,121). In an
accompanying experiment, root-cap cells of mung beans failed
to develop normally in microgravity (202); tissues were
poorly organized and cells appeared collapsed and degraded.
Furthermore, Moore et al. (156) demonstrated that decapped
roots of Zea mays L. failed to regenerate their caps in

5
xnicrogravity. Perturbed shoot and root orientation has been
observed repeatedly in plants grown in space (87) . And
lastly, changes in the structure of mitochondria,
dictyosomes, nuclei, and plastids have been noted in pea and
wheat plants grown in microgravity (87).
The physiology and development of microorganisms also
may be altered by microgravity (2,139,170,214,240). For
example, circadian rhythms in Neurospora crassa Shear &
Dodge (214) and Actinomyces levoris Kras (2), and the
development of fruiting bodies of the basidiomycete,
Polyporus brumalis (Pers.) Fris. (240), were disrupted in
microgravity. An increase in cell density was observed in
liquid cultures of Salmonella tvphimurium (Loeffler) Cast. &
Chai, exposed to microgravity, which indicated that the
space-flown cells grew faster than the ground controls
(139); a similar effect of microgravity on cell density was
not observed with Escherichia coli (Migula) Cast. & Chaim.
Although no studies on plant-microbe interactions in
microgravity have appeared in the literature, clinostats
(193) have been used to test the effects of gravity
compensation on plant disease development (48,89,119,232).
Gravity compensation has been reported to induce plant
responses similar to the effects of microgravity on plant
development (193). Curtis (48) reported that pustules of
the bean rust, caused by Uromvces phaseoli (Pers.) Wint.,
developed faster on gravity compensated beans compared to
nonrotated control plants. Wells and Baker (232) reported

6
that the fresh and dry weights of tumors induced by
Aorobacterium tumefaciens (Smith & Towns) Conn, were
significantly larger on carrot disks rotated on a clinostat
compared to tumors on nonrotated controls. These results
support the hypothesis that the severity of disease may be
greater in a microgravity environment than in a normal
terrestrial gravity. Although Brown et al. (25) cautioned
that all plants do not behave on clinostats exactly as they
behave in microgravity, the effects of microgravity on plant
(46,87,121,155,156,202) and microbial (2,139,170,214,240)
physiology support the hypothesis that new and unique host-
pathogen interactions may occur in a space-based CELSS.
Further research in microgravity phytopathology is obviously
required to determine the possible deletirious effects of
microorganisms on plant health.
It seems apparent that, because hydroponic plant
production systems are proposed for space-based CELSS
(30,101,124,166,219), an integrated pest management (IPM)
program might be developed for CELSS by applying available
information from terrestrial hydroponic systems. An IPM
program for CELSS will likely include components of
quarantine, sanitation, compartmentalization, plant
resistance, and constructed microbial communities (158,159).
In addition, ultraviolet radiation (35,67,210), heat
treatment (191), ozonation (190), ultrafiltrtation
(190,199), and bacterization (4,223,224) have proven
partially to moderately effective in suppressing root

7
diseases in terrestrial hydroponic systems and may be
applicable to CELSS. However, limitations in efficacy,
phytotoxicity, applicability, and energy requirements may
reduce the utility of these physical control options in
CELSS. Chemical biocides also have been proposed as pest
control options for space-based CELSS (219) and have proven
effective in terrestrial hydroponic systems for the control
of root diseases caused by Fusarium spp. (145,209,221).
However, strict air and water quality protocols for American
spacecraft (18) appear to preclude their use in CELSS.
Environmental manipulation of CELSS modules has not
been emphasized previously as an IPM component for disease
control, but it seems ideally suited for hydroponic plant
production systems in CELSS. Components of CELSS plant
production systems will be engineered for maximum
environmental controllability and compartmentalization to
optimize horticultural conditions for specific crops (124)
and to contain disease outbreaks (124,166). Temperature has
been used effectively to control root infection by Pvthium
aphanidermatum (Edson) Fitzp. and P. dissotocum Drechsler
(15,77) under hydroponic conditions. Furthermore, altering
the hydrogen ion concentration of the nutrient solution has
been reported to reduce the severity of disease caused by
Phvtophthora cinnamomi Rands (19) on avocado and Pvthium
debarvanum Hesse on lettuce (236).
Before initiating these experiments, the myriad of
possible pathosystems that could be studied in CELSS at

8
first appeared overwhelming. Indeed, if no studies have
been conducted on the effects of microgravity on host-
pathogen interactions, the study of any pathosystem might be
pertinent. However, it was decided that the selection of a
pathosystem should be based on the microbial contamination
reported from spacecraft, and that it should be based on the
occurrence of that microorganism as a significant pathogen
in terrestrial hydroponic systems. Pathogens in the genus
Fusarium fulfilled these two requirements. Fusarium spp.
have been isolated from Apollo spacecraft (96,180,181,218),
Shuttle payloads (144), and in experimental ground-based
CELSS (57,94,127). Furthermore, Fusarium spp. comprise the
second largest group of root pathogens in terrestrial
hydroponic systems (45,66,106,165,174,176,177,182,183,
184,212).
The first objective of this study was to test several
pathosystems under hydroponic conditions. The hosts were
chosen based on their selection as candidate plant species
for CELSS (101,219) and on their use in experiments in
microgravity (87,46,139,132). Fusarium solani (Mart.)
Appel. & Wr. f. sp. phaseoli (Burk.) Snyd. & Hans, and Viqna
radiata (L.) Wilczek were selected for subsequent
experiments because severe disease was observed in the
pathogenicity tests, and because pathogenesis appeared to be
influenced by temperature in a preliminary experiment (197).
The second objective of this study was to determine the
effects of temperature, hydrogen ion concentration,

9
humidity, and light quality on disease caused by F. solani
f. sp. phaseoli. These parameters are easily controllable
in closed systems (124,178,192) and might be used to manage
plant diseases in CELSS while avoiding the inclusion of
subsystems such as ultraviolet radiation, ozonation, or
ultrafiltration. Disease control using environmental
manipulation may have the additional advantage of being
adaptable to different plant production systems. In
contrast, subsystems like ultrafiltration or bacterization
might not be as widely applicable because of unique
constraints among different plant production systems. For
example, bacterization of plant roots growing in a
microporous tube system (56) might differ from bacterization
of roots in a NFT system because roots in the tube system,
unlike those in a NFT system, are partially exposed to air.
The third objective of this study was to determine the
effects of temperature and hydrogen ion concentration on the
attachment of macroconidia of F. solani f. sp. phaseoli to
roots of V. radiata. It was hypothesized that disease
control might be more effective by interfering with spore
attachment than by suppressing pathogenesis after plants
became infected.
The final objective of this study was to determine
whether spore attachment could be disrupted by the addition
of hapten sugars that were specific for plant lectins.
Lectins (phytoagglutinins) have been implicated in the
attachment of microorganisms to root surfaces (73,91,128,

10
129,130). Furthermore, a phytoagglutinin has been isolated
from hypocotyl cell walls (114,115,86), seeds (90), and
leaves (105) of V. radiata. Although no literature was
found that described phytoagglutinins from roots of V.
radiata, the presence of phytoagglutinins in roots of other
legumes (22,54,74,75,206) suggested that they may be
present. The activity of the phytoagglutinin from V.
radiata was shown to be sensitive to changes in hydrogen ion
concentration (114,115), and it seemed reasonable to suspect
that it might be involved with attachment of macroconidia of
F. solani f. sp. phaseoli.
In summary, these experiments were designed to
determine the effects of several environmental parameters on
disease caused by a pathogen that might pose a threat to
plant health in a space-based CELSS. Spore attachment by
the pathogen to roots was investigated because the process
was hypothesized to be affected by parameters of the
nutrient solution that are easily controlled in hydroponic
systems. Management of root diseases by altering the
environment in terrestrial hydroponic systems and in space-
based CELSS might be an inexpensive method for disease
control that could reduce construction costs of both
systems.

CHAPTER 2
EFFECTS OF CULTURE MEDIA ON MACROCONIDIUM MORPHOLOGY AND
PATHOGENICITY OF FUSARIUM SOLANI F. SP. PHASEOLI
Introduction
Cultural conditions can greatly affect colony
morphology, pigmentation, and sporulation of Fusarium spp.
on artificial media (69,149,160,168,204). Culture media
high in carbohydrates, such as potato dextrose agar (PDA),
can induce segmentation of cultures (160,230) or the
formation of patch mutants (149) with a corresponding
alteration, usually a decrease, in fungal pathogenicity or
virulence (7,31,148,160,168,203,230,231). Growth of
Fusarium spp. on PDA can also result in increased
variability of macroconidium morphology (160,168), but it is
unclear whether macroconidium morphology is linked to
differences of pathogenicity in the genus Fusarium. Natural
media were proposed by Snyder and Hansen (205) to reduce
these problems and enhance sporulation of fungi in culture.
Fisher et al. (69) and Nelson et al. (160) suggested
the use of carnation leaf agar (CLA) for the storage and
taxonomy of Fusarium spp. because of culture stability and
uniformity of macroconidium shape. However, CLA has been
employeed rarely for the production of inocula of Fusarium
spp. intended for pathogenicity tests. A review of papers
11

12
published in Phytopathology and Plant Disease, during the
periods of 1969 to 1989 and 1980 to 1989, respectively,
involving pathogenicity tests with Fusarium spp., indicated
that PDA was used for inoculum production in 43% of 244
studies; other artificial media rich in carbohydrates were
used in 19% of the studies; natural media (excluding CLA) or
naturally infested soil were used in 15% of the studies; and
various liquid media were used in 20% of the studies.
Carnation leaf agar was utilized in only 3% (7 of 244) of
the studies.
The objectives of this research were 1) to determine
the relative quality and quantity of macroconidia,
microconidia, and chlamydospores produced by isolates of
Fusarium spp. on different culture media; 2) to
quantitatively determine morphological differences among
macroconidia of Fusarium solani (Mart.) Appel. & Wr. f. sp.
phaseoli (Burk.) produced on two forms of PDA and two
natural media; and 3) to determine if there was variability
in pathogenicity or virulence of inoculum of F. solani f.
sp. phaseoli produced on different media. Portions of this
study were presented previously (197).
Materials and Methods
The identifications of Fusarium spp. were confirmed
with monoconidium cultures using the key of Nelson et al.
(83). The identification of F. solani f. sp. phaseoli was
based on pathogenicity tests (197) and on the key of Matuo

13
and Snyder (14 0) . Cultures of Fusarium spp. were maintained
in long-term storage at 4.0 C in a lyophilized form or on
slants of modified carnation leaf agar (MCLA), which is
described below. Short-term storage for specific
experiments was on MCLA.
Sterilization of leaves of carnation (Dianthus
carvophillus L., cv. 'Improved White Sim') and stem pieces
of mung bean (Vicma radiata (L.) Wilczek, cv. 'Berken') was
similar to the procedure of Fisher et al. (69). Plant
material was placed in capped, 250-ml glass jars and
irradiated with 15.1 megarads of gamma irradiation using a
Cobalt-60 source (Dept, of Chemistry, University of Florida,
Gainesville, FL, 32611). Irradiated samples were tested for
sterility by placing 30 randomly selected pieces of plant
material from each treated container on nutrient agar
composed of 3 g beef extract, 2.5 g glucose, 5 g peptone,
and 15 g of Bacto agar (Difco Laboratories, Detroit, MI,
48232) per liter of deionized water. Thirty pieces also
were placed on potato dextrose agar (Difco Laboratories)
supplemented with 1.0 ml of Tergitol NP-10 (Sigma Chemical
Co., St. Louis, MO, 63178) and 50.0 mg of chlortetracycline
hydrochloride (Sigma Chemical Co, St. Louis, MO, 63178)
(PDATC) per liter of medium (137).
Carnation leaf agar, prepared as was described by
Nelson et al. (160), consisted of irradiated carnation leaf
pieces placed on 2% water agar. Modified carnation leaf
agar was prepared by placing five to eight pieces

14
(approximately 3 mm x 5 mm) of irradiated plant material on
a nutrient solution agar (NSA) containing 2% Bacto agar and
the following inorganic salts (all obtained from Sigma
Chemical Co.): 4.51 mM Ca(N03)2•4H20, 3.48 mM KN03, 1.00 mM
KH2P04, 1.65 mM MgS04*7H20, 0.037 mM H3B04, 7.28 ¿iM
MnS04 *H20, 4.59 /xM ZnS04*7H20, 3.05 mM CuC12*2H20, 0.074 mM
(NH4)6 MO7024*4H20, and iron provided by equimolar
concentrations (89.5 nM) of FeCl3*6H20 and diethylene-
triaminepentaacetic. Hydroponic nutrient solutions used for
the following experiments were mixed fresh with
concentrations of ions identical to those used for NSA.
Potato dextrose agar was prepared as described by the
manufacturer (Difco Laboratories).
Forty-three isolates of Fusarium spp. (Table 2-1) were
evaluated for production of macroconidia, microconidia, and
chlamydospores on PDA, CLA, and MCLA. Macroconidia of
Fusarium spp. from MCLA cultures were streaked onto freshly
prepared PDA, CLA, or MCLA. Cultures were incubated for
14 days at 25 C under a 12-hr photoperiod. Light was
supplied by two, 20-watt, fluorescent bulbs at an intensity
— 9 — 1 . . • •
of 30 /zmol*m *s . Light intensity was measured with a
solar-IR light sensor (Campbell Scientific, Inc., Logan, UT,
84321) attached to an Omnidata Polycorder (Omnidata
International, Inc., Logan, UT, 84321). Cultures were first
studied without disturbing the agar surface using a stereo
microscope. Conidia, conidiophores, and chlamydospores were
then removed from each medium and stained in 0.05% trypan

Table 2-1. Production of macroconidia, microconidia, and chlamydospores by 43 isolates of
Fusarium spp. grown for 14 days on modified carnation leaf agar (MCLA), carnation leaf
agar (CLA), and potato dextrose agar (PDA).
Propagule typeP
Fusarium sod.
Number
of
isolates
Culture
media
Macroconidia
Microconidia
Chlamydospore:
F. culmorum
1*
MCLA
+++
VL
CLA
+++
-
VL
PDA
—
—
-
F. crraminearum
2^
MCLA
++
—
—
CLA
++
-
-
PDA
+
-
VL
F. lateritium
Is
MCLA
+
+++
—
CLA
+
+++
-
PDA
+
+++
-
F. moniliforme
2s
MCLA
+
+++
•
CLA
+
+++
-
PDA
+
+++
VL
F. oxvsoorum
5s
MCLA
+++
+
+++
CLA
+++
+
+++
PDA
VL
++
—
F. oxvsoorum f. sp.
lfc
MCLA
+
++
++
chrvsanthemii
CLA
+
++
++
PDA
++
+++
-

Table 2-1. Continued
Number
of Culture
Fusarium spp. isolates media
F. oxvsporum f. sp. 4U MCLA
conalutinans CLA
PDA
F. oxvsporum f. sp. lv MCLA
cucumerinum CLA
PDA
F. oxvsporum f. sp. MCLA
dianthi CLA
PDA
F. oxvsporum f. sp. 7r/w'x MCLA
lvcopersici CLA
PDA
F. oxvsporum f. sp. lr MCLA
melonis CLA
PDA
F. oxvsporum f. sp. 4^ MCLA
niveum CLA
PDA
F. oxvsporum f. sp. 7r,z MCLA
pisi CLA
PDA
Propagule type
Macroconidia Microconidia Chlamydospores
VL
+
+
VL
+
+
—
++
—
++
++
++
+++
++
++
VL
++
—
VL
+
+
VL
+
++
—
+++
—
++
+++
+
++
+++
+
++
+++
—
+++
++
+++
+++
++
++
+
++
VL
V
++
+
V
++
+
V
+++
—
V
V
V
V
V
V
V
V
V

Table 2-1. Continued.
Fusarium sdd.
Number
of
isolates
Culture
media
Propagule type
Macroconidia
Microconidia Chlamydospores
F. oxvsoorum f. so.
2X
MCLA
+
+
++
radicis-lvcopersici
CLA
++
+
++
PDA
+++
++
—
F. solani
2s
MCLA
+
+
++
CLA
+
+
++
PDA
+
++
—
F. solani f. sp.
lv
MCLA
+++
—
_
ohaseoli
CLA
+++
-
-
PDA
+++
—
—
F. solani f. sp.
lz
MCLA
VL
+
+
pisi
CLA
++
++
+
PDA
++
++
+
P + = propagule type observed, but not abundant; ++ = propagule type observed in moderate
abundance; +++ = propagule type observed at extremely high levels compared with other
cultures; - = propagule type not observed; V = presence or abundance of the propagule
type varied between different isolates of a particular Fusarium sp.; and VL = propagule
type observed, but in very low abundance.
^ Obtained from R. J. Cook, Washington State University, Pullman, WA, 99163.
r Obtained from J. J. Marois, University of California, Davis, CA, 95616.
® Obtained within The Land greenhouses, EPCOT Center, Lake Buena Vista, FL 32830.
Obtained from J. B. Jones, Gulf Coast Research and Education Center, Bradenton, FL
34203.

¡>i N
Table 2-1. Continued.
u Obtained from H. C. Kistler, University of Florida, Gainesville, FL 32611.
v Obtained from A. J. Anderson, Utah State University, Logan, UT 84322.
w Obtained from L. G. Brown, Division of Plant Industry, Gainesville, FL 32602.
x Obtained from J. P. Jones, Gulf Coast Research and Education Center, Bradenton, FL
34203.
Obtained from B. Larson, University of Florida, Gainesville, FL 32611.
Obtained from J. M. Kraft, United States Department of Agriculture, Agriculture Research
Service, Prosser, WA 99350.
00

19
blue (Eastman Kodak, Rochester, NY, 14650) in a lactic
acid:water:glycerol solvent (1:1:2). Further observations
were conducted under bright-field illumination with a Nikon
Optiphot compound microscope.
Pathogenicity tests were conducted on 19 pathosystems
with different cultivars of Phaseolus vulgaris L., Pisum
sativum L., Triticum avestivum L., and V. radiata
(Table 2-2). Seeds from each plant cultivar were germinated
in rolls of seed germination paper, as was described by
McClure and Robertson (141). Seven- to ten-day-old plants
of each cultivar were placed individually into five, 250-ml
flasks containing 225 ml of nutrient solution per flask and
grown under four, 40-watt, standard fluorescent bulbs at an
• . —9 —1
intensity of 100 jumol*m *s . Inoculum was prepared from
14-day-old MCLA cultures. Plants were inoculated at varying
densities between 350 and 8000 propagules/ml of nutrient
solution. The pH and temperature of the nutrient solution
were, respectively, 6.0-6.5 and 23-25 C at the time of
inoculation. Plants were maintained at room temperature
(approximately 23-25 C) for 14 days and then inspected for
root symptoms and wilt. Compressed air was passed through a
0.45-/m cartridge filter (Gelman Versaflow filter, Gelman
Sciences, Inc., Ann Arbor, MI, 48106) and distributed to
each 250-ml flask to aerate the nutrient solution.
Four pathosystems using F. solani f. sp. phaseoli
(Table 2-2) were selected for tests at 20, 25, and 30 C to
determine the effect of temperature on disease.

20
Table 2-2. Fusarium spp. tested for pathogenicity on
various hosts in a hydroponic system.
Fungus
Isolate
designation
Host
F. solani f.
ohaseoli
sp.
F-28AV
Phaseolus vulcraris
cv. Kentucky Wonder
cv. Pinto
cv. Red Kidney
Viana radiata
cv. Berken
F. solani f.
oisi
sp.
W8w
Pisum sativum
cv. Alaska
cv. Freezonian
cv. Tom Laxton
F. crraminearum
R2D-10-7X
Triticum avestivum
cv. Olsen's Dwarf
cv. Urguie
cv. Wampum
cv. Waverly
cv. Yecorro Rojo
F. culmorum
6A7 2AX
T. avestivum
cv. Olsen's Dwarf
cv. Yecorro Rojo
F. oxvsoorum
F-5^
T. avestivum
cv. Olsen's Dwarf
cv. Yecorro Rojo
F. oxvsoorum
f. so. oisi
Reiling-R2z
P. sativum
cv. Alaska
cv. Freezonian
cv. Tom Laxton
v Obtained from A. J. Anderson, Utah State University,
Logan, UT, 84322; isolate designation assigned at The
Land, EPCOT Center, Lake Buena Vista, FL 32830.
w Obtained from J. M. Kraft, United States Department of
Agriculture, Agriculture Research Service, Prosser, WA,
99350.
x Obtained from R. J. Cook, Washington State University,
Pullman, WA, 99163.
Â¥ Obtained from The Land greenhouses, EPCOT Center, Lake
Buena Vista, FL 32830.
z Obtained from J. J. Marois, University of California,
Davis, CA 95616.

21
Plants were grown in EGC model M-13 growth chambers
(Environmental Growth Chambers, Chagrin Falls, Ohio, 44022)
under conditions similar to those of the pathogenicity
tests, except that the photon flux was raised to 250-275
_ O _ *1
/¿mol-in **s . Seven plants per cultivar were used at each
temperature per each experiment. Inoculum of the pathogen
was prepared from a single isolate (F-28A) grown on MCLA for
14 days at 23-25 C. Plants were inoculated at a density of
500 propagules/ml. After 14 days plants were harvested and
the fresh and dry weights determined for each plant. The
experiment was conducted three times. A completely
randomized design was used to position replicates of each
treatment within growth chambers. The pathosystem of F.
solani f. sp. phaseoli and V. radiata. cv. 'Berken,' was
selected for further experiments because plant disease was
responsive to temperature changes and other pathosystems did
not consistently develop disease.
In a separate series of experiments, four different
media were used to produce macroconidia of F. solani f. sp.
phaseoli for studies on spore morphology and plant
pathogenicity. Preparation of PDA and MCLA was described
above. Fresh potato dextrose agar (FPDA), prepared as was
described by Nelson et al. (160), consisted of a potato
broth solidified using 1.5% Bacto agar; 2.0% dextrose was
added to the unsolidified potato broth. Mung bean stem agar
(MBSA) was prepared by placing 10-12, 1-cm-long pieces of
gamma-irradiated mung bean stems (V. radiata, cv. 'Berken')

22
on NSA. Macroconidia were streaked onto freshly prepared
MCLA, MBSA, PDA, and FPDA.
To determine the morphological differences among
macroconidia of F. solani f. sp. phaseoli produced on the
four different media, macroconidia were separately collected
from each medium using a platinum-wire loop and submersed in
20.0 n1 of 0.05% trypan blue stain for 5 min. Three
microliters of the stained macroconidium suspensions were
transferred to NSA and glass coverslips placed over the
microdrops. Agar blocks with the spores and coverslips were
excised from petri dishes and placed on glass microscope
slides. Macroconidia were observed using a Nikon Optiphot
microscope electronically coupled to a MicroComp® Integrated
Video Image Analysis System (Southern Micro Instruments,
Inc., Atlanta, GA, 30339). The length and width of 30,
randomly selected macroconidia from each sample were
measured. Five spore samples were prepared from separate
cultures in each of three experiments. In preliminary
tests, the accuracy of repeated measurements using the
MicroComp® Integrated Video Image Analysis System was
determined to be within 2% of the mean. The length of each
macroconidium was measured as depicted in Figure 2-1A. The
width of each macroconidium was measured at the widest point
of the spore. The procedure of mounting the stained
suspension of conidia on an agar surface was essential for
accurate measurements. Macroconidia would come to rest on
their lateral sides permitting precise measurement of each

Figure 2-1. Comparison of macroconidia of Fusarium solani
f. sp. phaseoli mounted on nutrient solution agar and in a
lactic acid, water, and glycerol solvent. A, Macroconidia
mounted in 0.05% trypan blue stain on nutrient solution agar
were measured in short straight lines to accommodate the
camber of the spore. B, Macroconidia mounted in trypan blue
stain or solvent were not accurately measured due to severe
differences in the focal planes between portions of
individual conidia.

24

25
conidium. When macroconidia were mounted on glass slides in
trypan blue stain or clear solvent, they were randomly
distributed at various angles (Figure 2-IB); individual
macroconidia would often fail to settle within a single
focal plane, and, thus, were very difficult to measure
accurately. Photographs were taken using Polaroid® 4x5
Instant Film, Type N55 (Polaroid Corp., Cambridge, MA,
02139). Data were analyzed using the Statistical Analysis
System® (SAS) (SAS Institute, Inc., Cary, NC, 27512).
Treatments were compared for equality in an Analysis of
Variance followed by Fischer's Least Significant Difference
Test (169) (P = 0.05).
A hydroponic system was used to test the pathogenicity
and virulence of inocula of F. solani f. sp. phaseoli
produced on PDA, FPDA, MCLA, and MBSA. Root temperature was
controlled at 20.0 ± 0.5 C using a closed-loop tube system
connected to a Lauda® RMS-20, chilled-water, recirculating
bath (Brinkman Instruments, Inc., Westbury, NY, 11590). A
single, 30-cm-long coil of 3.5-mm-diameter, stainless steel
tubing was placed into each plastic container and connected
to the main reservoir of the water bath by clear plastic
tubing (Nalgeneâ„¢ 8000 tubing, Nalgene Co., Rochester, NY,
14602). Experimental units consisted of 4-liter, plastic
containers wrapped on the sides with closed-celled foam,
duct-insulation tape (Macklenburg-Duncan, Oklahoma City, OK,
73118). Light was excluded from the nutrient solution by
the foam wrapping on the sides of the plastic containers and

26
by foam or black tape on the upper surface of the container
lids. The pH of the nutrient solution in each plastic
container was adjusted to 6.0 ± 0.2 units every 24 hr using
0.01 N HNO3 and 0.02 N KOH. A completely randomized design
was used to assign treatments to experimental units.
The following methods were tested for their utility in
producing seedlings of V. radiata: a slant-board culture
method (116), a method using rolls of seed germination paper
(141), a growth pouch method (31), and germination of seed
in 20-mesh silica sand or vermiculite. Problems were
encountered with these procedures that reduced the quality
of seedlings. Root hairs adhered to seed germination paper
in the first three procedures, resulting in root damage when
plants were transplanted. Branching characteristics of V.
radiata in all five methods were not similar to root
branching characteristics observed in hydroponic solutions.
A new technique was developed that minimized root contact
with solid materials and optimized root contact with the
hydroponic solution.
Seed of V. radiata. cv. 'Berken,' were surface steril¬
ized for 15 min in 0.13% NaOCl (2.5% bleach, Kare Chemical
Co, Opalocka, FL, 33054) plus 1.0 ml of Tween-20 (J.T. Baker
Chemical Co., Phillipsburg, NJ, 08865) per liter of
solution. Seed were rinsed three times in sterile deionized
water, allowed to imbibe water for 12 to 18 hr, and sown in
a seed germination tray (Figure 2-2) modified from a system
described by Prince and Knott (178). Plant supports of the

27
Figure 2-2. Schematic drawing of a cross-section through a
seed germination tray (t) used for the production of
seedlings of Viona radiata. Extruded plastic struts (ps)
were assembled by joining overlapping sections with two-part
epoxy (ep). A single layer of fiber-reinforced plastic
sheeting (p) was cut to fit through gaps between plastic
struts and extended low enough to be submersed 1.0-1.5 cm
below the surface of the nutrient solution (ns). Seed
germination paper (gp) was cut and fit over the plastic
sheeting to function as a wick for the nutrient solution.
Seed (s) were placed with their hilums down between opposing
surfaces of adjacent sections of seed germination paper.
Saran wrap (sw) was placed over the seed germination tray
for 3 days to enhance seed germination and hypocotyl (h)
extension. Cotyledons (c) would often dislodge from the
stem before transplanting seedlings. Root (r) growth
consisted primarily of first- and second-order roots.

28
seed germination trays were constructed of extruded plastic
struts, which were assembled in a series of slats using
common two-part epoxy. A single layer of fiber-reinforced
plastic sheeting was cut to fit through the gaps between the
plastic struts and then extended low enough to be submersed
1.0-1.5 cm below the surface of the nutrient solution. Seed
germination paper (no. 38 seed germination paper, regular
weight, Anchor Paper, St. Paul, MN 55101) was cut and fit
over the plastic sheeting to function as a wick for the
nutrient solution. Seeds were placed with their hilums down
between opposing sheets of seed germination paper (Figure
2-2). Plant supports were constructed to fit snugly into
28- x 38-cm polyethylene trays. Each tray was filled with
7.0 L of nutrient solution at pH 6.0-6.5.
Seed germination trays were covered with a single layer
of plastic saran wrap (Sealwrap, Borden Chemical, North
Andover, MA, 01845) to enhance seed germination and
hypocotyl extension, and placed under a combination of two,
400-watt, high pressure sodium lamps and two, 250-watt,
metal halide lamps (Energy Technics, York, PA, 17402).
Photon flux was 300 jumol *m-2 • s-1 at the top of seed
germination trays and was supplied in a 14-hr photoperiod.
The saran wrap was removed after 3 days and seedlings were
allowed to develop an additional 4-5 days. Root growth was
uninhibited and consisted of first- and second-order roots
(70) .

29
Five seedlings were transplanted into each plastic
container filled with 4.0 L of freshly mixed nutrient
solution. Each seedling was held in place by securing its
stem with a 28-mm-diameter, open-celled, foam plug (Dispoâ„¢
Plug, Baxter Healthcare Corporation, Stone Mountain, GA,
30083). Since a previous report suggested that open-celled
polyurethane foams can release phytotoxic volatiles and
should be heat or chemically treated prior to use (233),
foam plugs were heated at 70 C for 48-96 hr and then
autoclaved at 121 C and 1.1 kg*cm-2 for 25 min prior to use.
The nutrient solution in each plastic container was
aerated with filter-sterilized air using a 0.45-/nm cartridge
filter (Gelman Versaflow filters, Gelman Sciences, Inc., Ann
Arbor, MI, 48106) to maintain dissolved oxygen near
saturation (9.0-9.2 mg*L_1 at 20 C). Plants were grown
under eight, 1500-milliamp, Power Groove® fluorescent lamps
(General Electric Co., Cleveland, OH, 44112) which supplied
250-275 /¿mol-m-2 • s-1 at the top of the plant canopies.
The seed germination trays and experimental equipment
were assembled in a 2.5-m x 4.5-m research laboratory.
Ambient temperature was controlled by thermostatically
adjusting room temperature. Ambient humidity was provided
by a Through-Wall Humidifier (Model 707-TW, Hermidifier,
Inc., Lancaster, PA, 17603). Ambient temperature and
humidity for these tests were 21.3 ± 1.5 C and 60.9% ± 8.0%
relative humidity, respectively. Nutrient solution tempera¬
ture, ambient temperature, and ambient humidity data were

30
collected using the Campbell Scientific CR7X Measurement and
Control System (Campbell Scientific, Inc., Logan, UT, 84321)
and processed using The Land's Database Management System,
as has been described previously (157).
Inocula were prepared from 14-day-old cultures of PDA,
FPDA, MBSA, and MCLA. Plants were inoculated by adding
50 ml of a macroconidium suspension to produce a final spore
density of 500 propagules/ml of nutrient solution. Plants
were allowed to equilibrate in the plastic containers for
24 hr prior to inoculation. Two replicates per treatment
were inoculated in each experiment; two noninoculated
replicates served as controls. A completely randomized
design was used to assign treatments to experimental units.
The experiment was conducted three times. Fresh weights and
dry weights of individual plants were measured 14 days after
inoculation. The mean plant weight per experimental unit
was calculated, and data were subjected to an Analysis of
Variance to test for overall treatment equality, and
subsequently to Fischer's Least Significant Difference tests
(169) (P < 0.05).
Results
Isolates of the Fusarium spp. tested varied in
appearance between pinnotal forms and mycelial forms as has
been described by Nelson et al. (160). Patch mutants (149)
were observed on PDA with all isolates of F. solani,
F. culmorum (Smith) Sacc. , F. crraminearum (Schwabe) Snyd. &

31
Hans., and F. lateritium Nees emend. Snyd. & Hans., but were
observed less frequently with isolates of F. oxvsporum
(Schlect) Snyd. & Hans. Patch mutants occurred rarely on
MCLA and CLA. Sporodochia and phialides were present on the
wild-type mycelium of most isolates, but they were lacking
in patch mutants. Most isolates of Fusarium spp. produced
aberrant macroconidia and reduced quantities of
chlamydospores on PDA, as compared to the formation of these
structures on MCLA and CLA (Table 2-1). Aerial mycelium was
suppressed in most cultures grown on MCLA, when compared to
mycelium on CLA. Production of chlamydospores was
dramatically greater on both MCLA and CLA in cultures with
agar surfaces disturbed, such as by incisions from a
scalpel, than in undisturbed cultures. Macroconidia were
primarily produced on carnation leaf pieces on MCLA and CLA,
while chlamydospores were observed exclusively below the
agar surface.
In the preliminary pathogenicity tests, symptoms failed
to develop on T. avestivum inoculated with F. culmorum and
F. oxvsporum. even at the highest inoculum density of 8000
propagules/ml of nutrient solution. Root necrosis was
observed on all of the wheat cultivars when plants were
inoculated with F. qraminearum at 500 propagules/ml;
greatest disease occurred on cultivars Wampum and Urquie.
However, plant wilt did not occur with any of the wheat
cultivars inoculated with F. qraminearum. at any inoculum

level, nor was there any dramatic difference in vegetative
growth between inoculated and noninoculated plants.
32
Root symptoms developed on the pea cultivar Alaska, but
only when the inoculum density of F. solani f. sp. pisi
(Jones) Snyd. & Hans, exceeded 5000 propagules/ml. Fusarium
oxvsporum f. sp. pisi (Van Hall) Snyd. & Hans, induced root
symptoms on all three cultivars of P. sativum, but the
cultivar Freezonian was the least resistant. Plant wilt did
not occur in any pea cultivar inoculated with F. oxvsporum
f. sp. pisi.
Bean cultivars inoculated with F. solani f. sp.
phaseoli developed severe root necrosis that resulted in
either plant wilt or reductions in vegetative growth of the
host. In subsequent studies on the effects of temperature
on disease, 20-52% of P. vulgaris or V. radiata plants
wilted between 10 and 14 days after inoculation at 20 C.
Greatest plant damage occurred with the mung bean cultivar
Berken; 52% of plants wilted at 20 C and 21% of the plants
wilted at 25 C. Plant wilt did not occur at 30 C with any
of the bean cultivars. Further studies on the effects of
temperature on disease caused by F. solani f. sp. phaseoli
in V. radiata are presented in Chapter 3.
Distinct morphological differences were observed among
macroconidia produced on the different media. Macroconidia
produced on MCLA and MBSA (Figures 2-3A and 2-3B) stained
darker with trypan blue and had greater internal detail of
the cells than macroconidia produced on PDA and FPDA

Figure 2-3. Comparison of macroconidia of Fusarium solani f.
sp. phaseoli macroconidia produced on four different culture
media. Macroconidia were grown, harvested, stained, and
photographed, and negatives were printed, in identical
manners; thus, differences in size, cell-content complexity,
staining qualities, septation, and spore uniformity are
comparable. The dimension bar is applicable to all images.
Macroconidia produced on modified carnation leaf agar (MCLA)
(A) stained darker than those in other treatments.
Macroconidia produced on mung bean stem agar (MBSA) (B) were
similar to macroconidia produced on MCLA. Macroconidia
produced on MCLA and MBSA showed greater detail in their
cell contents than macroconidia produced on Difco potato
dextrose agar (PDA) (C) or fresh potato dextrose agar (FPDA)
(D). Macroconidia produced on PDA and FPDA were similar to
each other; they exhibited swollen and distended terminal
and intercalary cells, showed reduced staining
characteristics compared to macroconidia grown on MCLA and
MBSA, and had increased nonuniformity of conidium walls.

34
ÍvSíH

35
(Figures 2-3C and 2-3D). Septa were sharply delineated and
thicker in macroconidia produced on MCLA and MBSA, whereas
septa were not easily observed in macroconidia produced on
PDA and FPDA. Nonuniform and misshapen macroconidia were
produced in cultures of PDA and FPDA. Terminal and foot
cells of macroconidia were periodically distended (Figures
2-3C and 2-3D); swollen intercalary cells of macroconidia
were also observed. Severe distortions occurred on a very
low percentage of macroconidia produced on PDA and FPDA.
The predominant morphological distortion present on
macroconidia produced on PDA and FPDA was nonuniformity of
the side-walls of each conidium. Misshapen or distorted
macroconidia were not observed in MCLA or MBSA cultures.
The lengths and widths of macroconidia produced on all
media were distinctly different from each other (Table 2-3).
Macroconidia produced on PDA and FPDA were shorter and wider
than macroconidia grown on MCLA and MBSA (P < 0.05).
Furthermore, the variability of length and width was greater
for macroconidia produced on PDA and FPDA than for those
produced on MCLA and MBSA.
In the present study, distinct morphological
differences were observed in macroconidia produced on
different media. However, no differences in pathogenicity
or virulence were observed in tests with the pathosystem of
F. solani f. sp. phaseoli and V. radiata using macroconidia
produced on PDA, FPDA, MCLA, and MBSA (Table 2-4). Fresh

36
Table 2-3. The length and width of macroconidia of Fusarium
solani f. sp. phaseoli grown on four different media.
Media
Length (/m)
Width (|im)
Mean Range
Mean Range
Potato dextrose
agar
52.5
a
Fresh potato
dextrose agar
56.0
b
Modified carnation
leaf agar
59.8
c
Mung bean stem
agar
61.9
d
31.0-70.5
5.0
a
3.1-9.2
37.8-75.0
4.7
b
3.1-8.0
49.8-75.6
4.6
c
3.2-5.9
49.1-74.4
4.6
c
3.2-6.0
z Table values represent the means of 15 replicates (five
replicates per each of three experiments). Data were
analyzed using an Analysis of Variances followed by
Fischer's Least Significance Difference test. Values
followed by the same letter were not different (P > 0.05).

37
Table 2-4. Effects of culture media on the virulence of
inoculum of Fusarium solani f. sp. phaseoli on disease in
Viana radiata.
Treatment
Plant
fresh weight
(g)
Plant
dry weight
(g)
Percent
plant
wilt^
Noninoculated
control
4.88 a?
0.42 a
0 az
Potato
dextrose agar
1.54 b
0.18 b
83 b
Fresh potato
dextrose agar
1.79 b
0.22 b
67 b
Modified
carnation
leaf agar
1.77 b
0.21 b
77 b
Mung bean
stem agar
1.51 b
0.18 b
83 b
Â¥ Table values represent means of six replicates (two
replicates per each of three experiments). Data were
analyzed using Analysis of Variance and Fisher's Least
Significant Difference test; values followed by the same
letter were not different (P > 0.05).
z Data presented as percentages of plant wilt were
transformed to arcsines of square roots and then subjected
to an Analysis of Variance test followed by Fisher's Least
Significant Difference test; values followed by the same
letter were not different (P > 0.05).

38
weights and dry weights of plants receiving inoculum
produced on the different media were not different from each
other, but were different from noninoculated control plants
(P < 0.05). Plant wilt was also similar among the
treatments receiving inoculum.
Discussion
Macroconidium and colony morphology of Fusarium spp.
can be altered by ultra-violet light (148), differences in
wide-spectrum light quality (204), and cultural conditions
(69,148,149,160,168,194). Results of the study with 43
isolates of Fusarium spp. grown on PDA, CLA, and MCLA
indicate that variations in macroconidium morphology
produced on PDA occurred in a wide range of species in the
genus Fusarium. Variations in length and width, and the
development of swollen intercalary cells, were observed in
macroconidia produced on PDA. In contrast, macroconidia
produced on MCLA and CLA exhibited few distortions and were
of uniform size. In addition, the relative abundance of
macroconidia, microconidia, and chlamydospores from Fusarium
spp. grown on PDA, MCLA, and CLA were different. The width
and length of macroconidia, of at least a few Fusarium spp.,
have been shown to be altered by production on media with
high carbohydrate concentrations (69,136,168,194). Marchant
and White (136) reported swollen intercalary cells in
macroconidia of F. culmorum that were grown in the presence
of glucose and a nitrogen source. Macroconidia of F. solani

39
f. sp. phaseoli also developed swollen intercalary cells
when they were incubated in 50-mM solutions of glucose,
sucrose, and mannose (Chapter 6). Spore swelling was not
observed when macroconidia were allowed to germinate on root
surfaces (Chapter 4). Thus, it appears that morphometric
changes of macroconidia may represent artifacts induced by
the production or incubation of spores in environments high
in carbohydrates.
Morphological characteristics of macroconidia are
critical for current taxonomic systems in the genus Fusarium
(20,160,218), and the need for standardization of cultural
conditions for taxonomic purposes (160) is generally
accepted. However, the usefulness of macroconidium
morphology for predicting the pathogenicity of cultures of
Fusarium spp. is guestionable. Although previous workers
have demonstrated that cultural variability of Fusarium spp.
induced on media high in carbohydrates can correlate to
alterations in pathogenicity (7,69,148,160,194,230,231), few
studies have concomitantly investigated morphological and
pathological differences between macroconidia produced on
different media (80,194,230,231). Oswald (168) identified
spore width as the primary difference among culture variants
of F. culmorum. F. equiseti (Corda) Sacc., and F.
graminearum. but did not correlate changes in spore
morphology to changes in pathogenicity. Sano and Ui (194)
reported that pathogenicity and macroconidium length were
less, and macroconidium width greater, when inoculum of F.

40
solani f. sp. phaseoli was produced on a medium high in
carbon. Morphometric differences in the length and width of
macroconidia of F. solani f. sp. phaseoli produced on the
high carbon-containing media PDA and FPDA were consistent
with the study of Sano and Ui (194), but in the current
study, no differences were observed in pathogenicity with
inoculum produced on MCLA, MBSA, PDA, and FPDA.
The severity of disease caused by F. solani f. sp.
phaseoli in V. radiata (Chapter 3) and spore attachment of
the pathogen to roots of the host (Chapter 4) have been
shown to be affected by temperature and hydrogen ion
concentration of the nutrient solution. It has yet to be
demonstrated whether these environmental factors alter the
virulence of Fusarium spp. grown on different media.
Differences between the results from the current study and
those of Sano and Ui (194) might be due to different plant
production methods, culture media utilized for the
production of inoculum, or temperature and hydrogen ion
effects on spore attachment or virulence of inoculum.
It is not clear whether Sano and Ui (194) maintained
cultures of F. solani f. sp. phaseoli by repetitive
transfers on media with a high carbon concentration.
Previous studies have shown that repetitive transfers on
high carbon-containing media could induce increased
morphological variability of macroconidia (69,160,168,194)
and alter the pathogenicity of F. solani f. sp. phaseoli
(148,160,168,203). In the present study, inoculum was

41
prepared by transferring spores from cultures of MCLA, a low
carbon-containing medium, to media with either high or low
carbon; the effects of repetitive transfers with each medium
on pathogenicity were not evaluated.
Although it is unclear whether macroconidium morphology
is linked to differences in pathogenicity in the genus
Fusarium, culture variability, notably between the pinnotal
colony type and mycelial colony type, has been correlated to
modifications in pathogenicity (69,160,168,230,231).
Wellman and Blaisdell (230,231) described five basic colony
forms produced by F. oxvsporum f. sp. lvcopersici (Sacc.) on
different media that correlated to consistent differences in
pathogenicity. Results from the current study do not
support the conclusion that macroconidium variability
correlates to differences in fungal pathogenicity.
Additional research is required before any comprehensive
conclusions can be drawn on the concomitant effects of
culture media on spore morphology and fungal pathogenicity
in the genus Fusarium.

CHAPTER 3
EFFECTS OF TEMPERATURE, HYDROGEN ION CONCENTRATION,
HUMIDITY, AND LIGHT QUALITY ON DISEASE CAUSED BY FUSARIUM
SOLANI F. SP. PHASEOLI IN VIGNA RADIATA
Introduction
Controlled ecological life support systems (CELSS) have
been proposed for space bases to regenerate oxygen, water,
and food from human and industrial wastes (18,133,166).
Biological processes will be combined with physical and
chemical technologies to provide life support systems that
will reduce requirements for the resupply of materials from
Earth. Higher plants are considered the primary candidates
for photosynthesis in CELSS (101,166,178,219) and various
methods of plant production have been proposed
(30,56,166,178,235). Hydroponic plant production systems
appear to have several advantages over lunar regolith (30)
and other solid substrates (166).
Plants in CELSS may be threatened by microorganisms
transported into space as contaminants on spacecraft
components, equipment, astronauts, and plant-propagative
materials. Extensive microbial contamination has been
reported in American spacecraft (24,96,131,144,180,181,
215,217,218,228). Species of Alternaria. Aspergillus.
Candida. Cephalosporium. Cladosporium. Fusarium. Mucor,
Penicillium. Phoma. Saccharomvces. and Trichoderma were
42

43
among the most prevalent fungal contaminants of these
systems. Predicting deletirious plant-microbe interactions
in space-based CELSS is hampered by an extreme paucity of
information on plant-microbe interactions in space.
However, because hydroponic plant production systems will be
utilized in space-based CELSS (30,101,124,166,219), it seems
likely that an integrated pest management (IPM) program
might be developed by applying available information from
terrestrial hydroponic systems.
In terrestrial environments, various plant pathogens
have been described as significant problems in hydroponic
systems. Species of Pvthium and Phvtophthora have been the
most frequently reported root pathogens in terrestrial
hydroponic systems (15,29,66,72,77,100,117,175,198,208,210,
211,225). However, species of Pvthium and Phvtophthora have
not yet been detected as contaminants in American spacecraft
and should be avoidable in space-based CELSS through strict
sanitation and quarantine procedures.
Fusarium spp. have been isolated from Apollo spacecraft
(96,180,181,218), shuttle payloads (144), and in
experimental ground-based CELSS (57,94,127). Furthermore,
Fusarium spp. comprise the second largest group of root
pathogens in terrestrial hydroponic systems (45,66,106,165,
174,176,177,182,183,184,212,226). Couteaudier and
Alabouvette (page 153, cit. 45) concluded that wilt diseases
caused by Fusarium spp. were ". . .the most important
pathological problem in soilless culture.” Nowicki (165)

44
found that formae speciales of Fusariuxn oxysporum
(Schlecht.) and Fusarium solani (Mart.) Appel. & Wr.
constituted 51% (213 of 415 isolates) of seven Fusarium spp.
identified as pathogens of greenhouse-grown cucumbers during
a 5-year survey in Poland. Based on this literature, it
seems apparent that Fusarium spp. will likely occur as
contaminants in space-based CELSS, and when present they may
constitute a threat to plants.
An integrated plant disease control program for CELSS
will likely include components of quarantine, sanitation,
compartmentalization, plant resistance, and constructed
microbial communities (158,159). Environmental manipulation
of CELSS modules has not been emphasized previously as an
IPM component for disease control, but seems ideally suited
for hydroponic plant production systems in CELSS. Although
temperature has been used effectively to control root
infection by Pvthium aphanidermatum (Edson) Fitzp. and P.
dissotocum Drechsler under hydroponic conditions (15,77),
few other studies have been found that explore the area of
environmental manipulation for disease control in hydroponic
systems (95,117,207).
The objectives of this study were 1) to test the
effects of temperature, hydrogen ion concentration,
humidity, and light quality on disease caused by F. solani
f. sp. phaseoli on Viona radiata (L.) Wilczek under
hydroponic conditions; 2) to determine the fluctuations in
nutrient solutions of primary and secondary inoculum of

45
F. solani f. sp. phaseoli under different environmental
conditions; 3) to assess whether plant growth-inhibiting or
growth-promoting compounds accumulated in nutrient solutions
during experiments on the environmental effects on disease;
and 4) to determine if severe root disease contributes to
yield reductions in V. radiata by inducing plant nutrient
deficiencies. Viana radiata was chosen for these studies
based on its selection as a candidate crop species for CELSS
(219) and its sensitivity to F. solani f. sp. phaseoli under
hydroponic conditions (Chapter 2).
Materials and Methods
Experiments on temperature, hydrogen ion concentration
([H+]), and humidity were conducted in environmental plant-
growth chambers (Model M-13, Environmental Growth Chambers,
Inc., Chagrin Falls, OH 44022). Light for each growth
chamber was provided in a 12-hr diurnal cycle by 16, 1500-
milliamp, Power-Groove® fluorescent lamps (General Electric
Co., Cleveland, OH, 44112) and six, 25-watt, incandescent
bulbs. Florescent lamps were periodically changed, and
distances between the lights and plant canopies were
periodically adjusted to maintain a constant photon flux of
250 /¿mol• s-1 *m-2 at the tops of the plant canopies. Light
intensity for all experiments was measured with a Li-Cor,
LI-190SB, radiation sensor (Li-Cor, Ltd., Lincoln, NE,
68504) calibrated to measure spectral radiance between 400
and 700 nm.

46
Experiments on light quality were conducted in a 2.5-m
x 4.5-m, tissue culture clean-room. High intensity
discharge (HID) lamps were suspended over the plant-growing
surface from metal supports made of 2.54-cm aluminum tubing.
To prevent cross-exposure to light, HID lamps were separated
by suspending sheets of white plastic from the metal
supports. Complete light spectra of HID lamps were obtained
using a Li-Cor, LI-1800 spectroradiometer (Li-Cor, Ltd.,
Lincoln, NE 68504) calibrated to determine lamp output in 2-
nm bands between 330 and 1100 nm. Light was provided in a
12-hr diurnal cycle by the following HID lamps: two, 250-
watt, high pressure sodium (HPS) fixtures (Energy Technics,
York, PA, 17402); two, 400-watt, metal halide (MH) fixtures
(Energy Technics, York, PA, 17402); and eight, high
intensity fluorescent bulbs (FL) (Chapter 2) assembled in a
specially designed 91.4-cm x 121.9-cm fixture (New Horizons
Lighting, Palm City, FL, 34990). Light intensity from each
combination of lamps was measured weekly; as lamps aged
light intensity would drop by up to 20% with FL lamps and
between 5-10% with MH and HPS lamps. The distances from the
HID lamps to plant canopies were periodically adjusted to
maintain a constant photon flux of 250 ¿¿mol• s-1 *m-2 at the
tops of the plant canopies. Fluorescent lamps were changed
every 4 to 8 weeks to assist in maintaining the appropriate
light intensity. Plexiglass® barriers (Rohm & Haas,
Philadelphia, PA, 19105) were placed between all lamp

47
fixtures and plant canopies to reduce radiant and convective
heating of plant foliage.
Air circulation was provided in each HID lamp zone by
individual air blowers (Model 4C446 Dayton Electric Mfg.
Co., Chicago, IL 60648) connected to the outlet of the room
air conditioning system. Air temperature was maintained at
20 or 25 C, ± 2.0 C, by adjusting the room thermostat. Root
temperature control was within 0.5 C of set-points and was
provided by Lauda® temperature-control baths, as was
described previously (Chapter 2). Humidity was maintained
at a vapor pressure deficit (VPD) of 6.0 mm Hg using
individual humidifiers (Model 707-TW, Hermidifier, Inc.,
Lancaster, PA, 17603) in each HID lamp zone.
The following procedure for production of seedlings,
assembly of experimental units, inoculation of appropriate
treatments, and completion of each experimental repetition
was identical for all experiments and was similar to the
procedure described in Chapter 2. Experimental units
consisted of 4-L plastic containers wrapped in 5.0 mm of
foam insulation tape (Chapter 2). Eight plastic containers
were arranged in each HID lamp-zone or growth chamber.
Five, 7- to 8-day-old seedlings of V. radiata, cv. 'Berken,'
were transplanted into each plastic container; plants were
held in place with the aid of 28-mm-diameter, foam plugs
pretreated by dry heat and autoclaved, as was described
previously (Chapter 2). A modified Hoagland's nutrient
solution (Chapter 2) was prepared fresh from reagent grade

48
salts and deionized water. All chemicals were obtained from
Sigma Chemical Co. (St. Louis, MO, 63178) . The pH of the
nutrient solution was maintained at 6.0 for the temperature,
humidity, and HID lamp experiments, but adjusted to the
appropriate [H+] during the experiments on the effects of
the [H+] on disease. The [H+] was adjusted in all
experiments using 0.01 N HN03 and 0.02 N KOH.
Inoculum of F. solani f. sp. phaseoli (isolate F-28A,
obtained from A. J. Anderson, Utah State University, Logan,
Utah) was prepared on a modified carnation leaf agar (MCLA)
(Chapter 2) and consisted exclusively of macroconidia.
Inoculum in the stock suspension in deionized water was
adjusted to a density of 5.6 x 104 propagules/ml. A
hemacytometer was used to count the numbers of propagules/ml
of solution in this and all subsequent experiments. The
inoculum density of the stock suspension was confirmed by
serial dilutions and a micro-drop counting procedure in
which 10, l-/xl samples from the inoculum stock suspension
were placed on the surface of nutrient solution agar (NSA)
(Chapter 2); the number of macroconidia in each microdrop
was then counted. Plants were inoculated in 50% of the
experimental units per HID lamp-zone or growth chamber by
injecting 50 ml of inoculum as five, 10-ml samples into each
plastic container.
To determine the fluctuations in the numbers of
propagules of F. solani f. sp. phaseoli in the nutrient
solution of each treatment, three, 1-ml samples were taken

49
from each of two experimental units per growth chamber or
HID lamp-zone at 0, 1, 5, 10, and 14 days after inoculation.
Each sample was dispensed onto five plates of potato
dextrose agar supplemented with 50.0 mg of chlortetracycline
hydrochloride and 1.0 ml of tergitol NP-10 per liter of
medium (PDATC) and incubated under low light (< 10 ¿¿mol*
s-1*m-2) for 5-7 days. Colonies of F. solani f. sp.
phaseoli were counted for each sample date; isolates of
other fungal species were obtained from these cultures and
identified to genus using available keys
(12,36,42,61,160,229) .
Plants were incubated for 14 days under various
combinations of temperature, [H+], humidity, and light
quality, as will be described below. The numbers of plants
exhibiting wilt symptoms at 14 days were counted for each
treatment. Shoots and roots were measured individually for
fresh and dry weights. Data were pooled and adjusted to
yield mean plant fresh and dry weights per each experimental
unit. Root samples were obtained from two plants per
experimental unit to visually determine the presence of
macroconidia and conidiophores produced on root surfaces.
Root samples were taken between 7 and 14 days after
inoculation from inoculated plants maintained at 20 C and
from all treatments at 14 days. Root samples were stained
in 0.05% trypan blue (Chapter 2) and viewed at various
magnifications with a Nikon Optiphot compound microscope.
The morphometric method of root analysis described by Fitter

50
(70) was utilized throughout these experiments. To deter¬
mine the percentage of roots colonized by F. solani f. sp.
phaseoli. three, 1.0-cm-long root segments were excised from
symptomatic and asymptomatic roots from both noninoculated
and inoculated treatments. Root segments were placed on
PDATC and incubated under low light for 5-7 days.
Root segments were prepared for scanning electron
microscopy (SEM) by fixation in phosphate buffered 3%
glutaraldehyde and 1% osmium tetroxide (Chapter 5). Samples
were dehydrated in ethanol and critical-point-dried with
CC>2. Specimens were mounted, coated with gold, and viewed
with a Hitachi S-530 scanning electron microscope, as
described in Chapter 5.
A bioassay was developed to determine if plant growth-
promoting or growth-inhibiting compounds, that could have
contributed to differences in plant weights among
treatments, had accumulated in the nutrient solution of
various treatments. The bioassay was modified from the
procedures of Bentley and Bickle (16). Two hundred
milliliters of nutrient solution were collected from each
experimental unit of both noninoculated and inoculated
treatments. Samples were bulked for each treatment and
filter-sterilized through 0.45-jum, Gelman Mini-Capsule,
cartridge filters (Gelman Sciences, Inc., Ann Arbor, MI,
48106). Bulked samples were stored at 4 C until used in the
bioassays.

51
For each bioassay sample tested, 100 ml of nutrient
solution (pH 6.0) containing 3 g Bacto agar (Difco
Laboratories, Detroit, MI, 48232) were placed in a 500-ml
flask and autoclaved for 20 min at 1.1 kg*cm-2 and 240 C.
The agar was allowed to cool to 60 C, and 100 ml of test
nutrient solution from an individual treatment were added to
the flask; agar was poured immediately into two, square
polystyrene petri dishes. Prior to the addition of the
bioassay sample to the autoclaved agar, it was adjusted to
pH 6.0 and sterilized a second time using a 0.45-/¿m,
cellulose nitrate filter and Nalgene® PSF filtration system
(Baxter Healthcare Corp., McGraw Park, IL, 60085). A
separate sample of NSA was prepared from 200 ml of fresh
nutrient solution to serve as a nonexperimental control in
the event that both inoculated and noninoculated treatments
were similar in their effects on germinating seed.
Seeds of Lactuca sativa L., cv. 'Great Lakes,' were
surface-sterilized for 15 min with intermittent agitation in
a solution of 0.13% NaOCl (2.5% bleach, Rare Chemical Co.,
Opalocka, FL, 33054) with Tween-20 (J.T. Baker Chemical Co.,
Phillipsburg, NJ, 08865) added at the rate of 1.0 ml*L-1.
Seeds were rinsed three times in sterile deionized water and
allowed to imbibe water for 6-8 hr. Twenty-five seeds were
placed on each of two plates of bioassay media prepared from
each treatment. In preliminary studies, the lettuce
cultivar Great Lakes was selected based on its sensitivity

52
to 2,4,5-triphenyl-2H-tetrazolium chloride at a concentra¬
tion of 0.05 mg*L-1 (data not shown).
Plates were wrapped with parafilm and incubated at 25 C
in a Percival I-30BLL incubator (Percival Manufacturing Co.,
Boone, IA 50036) for 4 days for the temperature experiments
and 3 days for the [H+], humidity, and light quality
experiments. A 12-hr, diurnal light cycle was provided
using four, 20-watt, fluorescent bulbs, which yielded a
photon-flux of 50 /Limol*s *m . All bioassay plates were
inclined 45° during incubation. After incubation, root tips
were marked and the lengths of the seedling radicles were
measured using a Sony DXC-3000 color video camera (Sony
Corporation, Japan) electronically coupled to the Microcomp®
Integrated Video Image Analysis System (Southern Micro
Instruments, Inc., Atlanta, GA, 30339), as was described
previously (Chapter 2).
Statistical analyses were conducted with the
Statistical Analysis System (SAS) (SAS Institute, Inc.,
Cary, NC, 27512). Unless otherwise stated, statistical
significance was at the P < 0.05 level. Square-root or log
transformations were utilized in most experiments to induce
homogeneity of variances among treatments. A 0.25-power
transformation was appropriate for a few experiments because
square-root and log transformations, respectively, either
under- or over-transformed the data. Plots of residuals and
predicted values were used to determine the appropriate
transformation for each experiment (213). Orthogonal

53
polynomial contrast analyses were used when treatments were
equally spaced and quantitatively related (213); standard
Analysis of Variance was used for data that were not
quantitatively related. Least-squares mean separation tests
were employed where significant F-values indicated treatment
effects.
A split-plot design was used for the study on the
effects of temperature on disease; temperature (20, 24, 28,
32, and 36 C) was the main plot and inoculum level (0 and
500 propagules/ml) the split plot. Temperature was
replicated three times temporally, while inoculum level was
replicated four times within each repitition of the main
plot. The experimental mean for humidity, across all
temperatures tested, was 12.0 mm Hg VPD (50% relative
humidity at 25 C).
A split-split-plot design was used for the studies on
the effects of [H+], humidity, and light quality on disease.
In the experiments on [H+], temperature (20 and 25 C) was
the main plot, [H+] (pH 4.0, 5.0, 6.0, and 7.0) was the
split plot, and inoculum level (0 and 500 propagules/ml) was
the split-split plot. Temperature, inoculum level, and [H+]
each were replicated twice, and each test within the
experiment was conducted three times. During these tests
humidity was maintained at a VPD of 6.0 mm Hg.
In the experiments on humidity, temperature (20, 25,
and 30 C) was the main plot, humidity (6.0 and 12.0 mm Hg,
VPD) was the split plot, and inoculum level (0 and 500

54
propagules/ml) was the split-split plot. Temperature,
humidity, and inoculum level each were replicated two times
temporally.
In experiments on the effects of light quality on
disease, temperature (20 and 25 C) was the main plot, HID
lamp type was the split plot, and inoculum level (0 and 500
propagules/ml) was the split-split plot. Temperature and
light quality each were replicated three times temporally,
while inoculum level was replicated four times within each
repitition of the main plot. During these tests humidity
was maintained at a VPD of 6.0 mm Hg.
Trifoliate leaves of V. radiata from experiments on the
effects of temperature, [H+], humidity, and light quality
were oven-dried at 70 C for 3-5 days. Replicate samples
from each treatment were bulked and then ground to pass a
40-mesh metal screen using a Whiley mill. Elemental
analysis was conducted by Dr. Wade Berry, University of
California, Berkeley, CA, using an optical emission
spectrometry system (3). Ion composition of trifoliate
leaves from experiments on the effects of temperature,
humidity, and [H+] on disease were analyzed using orthogonal
polynomial contrasts; data from the experiment on the effect
of light quality on disease were analyzed using Analysis of
Variance.

55
Results
Shoot weights, root weights, whole plant weights, and
shoot-root ratios were analyzed separately for each
experiment. Most treatment effects were consistent,
regardless of plant part analyzed; whole plant fresh weights
are presented for simplicity. Whole plant dry weights and
shoot-root ratios are presented in Appendix A.
Growth of noninoculated V. radiata plants was influenced by
temperature, but not by [H+], humidity, and light quality
(Tables 3-1, 3-2, 3-3, and 3-4). Optimum growth occurred
between 28-32 C (Table 3-1). Noninoculated plants grown at
20 C were approximately 30-35% as large as plants grown at
28-32 C (Tables 3-1 and 3-3).
The severity of disease caused by F. solani f. sp.
phaseoli in V. radiata was influenced by temperature (Table
3-1) and [H+] (Table 3-2), but not by humidity (Table 3-3)
and light quality (Table 3-4). Disease was greatest at 20 C
for all experiments (Table 3-1, 3-2, 3-3, and 3-4); between
52 and 97% of inoculated plants wilted by 14 days after
inoculation, and plant fresh weights were reduced an average
of 60-75% compared to noninoculated plants at 20 C.
Differences between noninoculated and inoculated plants at
24-28 C also were significant (P < 0.05). Weights of
noninoculated and inoculated plants at 30 C, or above, were
not different (P > 0.10) (Table 3-1 and 3-3). The effects
of temperature between 20 and 36 C (Table 3-1) were best
described by quadratic polynomial equations for

56
Table 3-1. Effects of temperature on disease caused by
Fusarium solani f. sp. phaseoli in Vigna radiata.
Temperature Plant fresh weight (g)
(C)
Noninoculated Inoculated
20
4.45
a2
1.76* a
24
8.58
b
6.73* b
28
13.44
d
11.11* d
32
13.71
d
13.40 e
36
10.48
c
10.84 c
2 Table values represent the means of 12 replicates per
treatment (four replicates per treatment in each of three
experiments). Data were transformed to logs and then
subjected to an orthogonal polynomial contrast analysis;
table values are presented as detransformed numbers.
Least-squares mean separation tests were used to examine
pairwise comparisons between appropriate treatments.
Significant differences in the comparisons between
noninoculated and inoculated plants at each temperature
are indicated by an asterisk (*) (P < 0.05). Treatments
in columns followed by the same letter were not different
(P > 0.05).

57
Table 3-2. Effects of hydrogen ion concentration and
temperature on disease caused by Fusarium solani f. sp.
phaseoli in Viqna radiata.
PH
Temperature
(C)
Plant fresh weight (g)
Noninoculated
Inoculated
4.0
20
4.23
az
1.52* a
25
9.99
b
9.74 c
5.0
20
4.36
a
1.08* a
25
10.90
b
8.28* be
6.0
20
4.61
a
1.25* a
25
10.49
b
8.49* be
7.0
20
4.44
a
1.07* a
25
9.78
b
7.13* b
z Table values represent the means of six replicates per
treatment (two replicates per treatment in each of three
experiments). Data were adjusted with a 0.25-power
transformation and then subjected to an orthogonal
polynomial contrast analysis; table values are presented
as detransformed numbers. Least-squares mean separation
tests were used to examine pairwise comparisons between
appropriate treatments. Significant differences in the
comparisons between noninoculated and inoculated plants
for each temperature and hydrogen ion concentration are
indicated by an asterisk (*) (P < 0.05). Treatments in
columns followed by the same letter were not different
(P > 0.05).

58
Table 3-3. Effects of humidity and temperature on disease
caused by Fusarium solani f. sp. phaseoli in Viqna radiata.
Humidity
(mm Hg, VPD)^
Temperature
(C)
Plant fresh
weight (g)
Noninoculated
Inoculated
6.0
20
4.98
az
1.62* a
25
13.44
b
9.61* b
30
15.18
b
15.77 c
12.0
20
4.78
a
1.72* a
25
13.62
b
9.76* b
30
14.92
b
14.96 c
y Humidity was measured as vapor pressure deficit (VPD) in
mm Hg; vapor pressure deficits of 6.0 and 12.0 mm Hg, at
25 C, correspond to 75% and 45% relative humidities,
respectively.
z Table values represent the means of eight replicates per
treatment (four replicates per treatment in each of two
experiments). Data were transformed to logs and then
subjected to an orthogonal polynomial contrast analysis;
table values are presented as detransformed numbers.
Least-squares mean separation tests were used to examine
pairwise comparisons between appropriate treatments.
Significant differences in the comparisons between
noninoculated and inoculated plants at each temperature
and humidity are indicated by an asterisk (*) (P < 0.05).
Treatments in columns followed by the same letter were
different (P > 0.05).

59
Table 3-4. Effects of light quality and temperature on
disease caused by Fusarium solani f. sp. phaseoli in Viana
radiata.
Lamp-type
Temperature
(C)
Plant fresh weight (g)
Noninoculated
Inoculated
Fluorescent
20
4.76
az
1.60* a
25
9.49
b
5.77* b
Metal halide
20
4.51
a
1.57* a
25
8.93
b
5.75* b
High pressure
20
5.09
a
1.71* a
sodium
25
11.16
b
7.53* b
z Table values represent the means of 12 replicates per
treatment (four replicates per treatment in each of three
experiments). Data were transformed to logs and then
subjected to an Analysis of Variance and a least-squares
mean separation test; table values are presented as
detransformed numbers. Significant differences in the
comparisons between noninoculated and inoculated
treatments at each temperature and lamp-type are indicated
by an asterisk (*) (P < 0.05). Treatments in columns
followed by the same letter were not different (P > 0.05).

60
noninoculated and inoculated plants (P < 0.001). Plant wilt
varied among experiments and was not considered a reliable
estimate of the severity of disease because at 25 C plants
periodically would recover from wilt by initiating lateral
roots from their lower stems just above symptomatic tissues.
Necrotic flecks were observed at 20 and 25 C on all
root orders 72-96 hr after inoculation. At 20 C, necrotic
flecks coalesced into larger necrotic lesions by 5 days, and
first- and second-order roots were moribund by 10 days after
inoculation. At 24 and 28 C, more severe symptoms developed
on second-order roots with root hairs than developed on
first- or second-order roots without root hairs. Lower
hypocotyls were less affected by F. solani f. sp. phaseoli
than the root systems; tissue necrosis usually did not
develop acropetally to plant crowns. Root symptoms were
most severe at 20 C and declined dramatically as temperature
increased; no root symptoms were observed on lateral roots
at 30 C or above. Small necrotic lesions were observed on
the main tap roots just below the crown at 30 C, but not at
32 and 36 C. When root necrosis occurred, it was most
severe in the crown area of plants, regardless of the
treatment parameter. At 24 and 28 C, plants initiated new
lateral roots from severely damaged tissues, presumably from
intact pericycle cells, between 10 and 14 days after
inoculation.
Conidiophores and macroconidia were produced between 10
and 14 days after inoculation at 20 C and by 14 days at 24

61
and 28 C. Conidiophores and macroconidia were most abundant
on heavily damaged roots maintained at 20 C; they were less
abundant as temperature increased to 28 C, and they were
absent from inoculated roots at 30-36 C. Highly branched
monophialides and sporodochia (Figure 3-1) were observed
between 20 and 25 C on second-order roots, but not at 28 C
and not on any other root orders. Branched monophialides
emerged directly from the root epidermis, root tips, and
from cortical cavities formed when longitudinal splits
occurred in the root epidermis on severely damaged roots
(Figure 3-1). At 20-28 C unbranched monophialides were
produced from individual hyphae on the root surface, termed
rhizosphere hyphae to differentiate them from hyphae
emanating from cortical cavities (Figure 3-1C).
Microconidia were not observed in the rhizosphere under any
environmental conditions; chlamydospores were only rarely
observed at 20 C. Development of branched and unbranched
monophialides, sporodochia, and secondary macroconidia was
consistent at each temperature, but was unaffected by light
quality and humidity. Mature conidiophores and macroconidia
of F. solani f. sp. phaseoli were not observed at pH 4.0;
sporodochia and branched monophialides were disorganized and
poorly developed (Figure 3-2). Mature conidiophores and
macroconidia were formed at pH 5.0, 6.0. and 7.0, at both 20
and 25 C.
At temperatures between 20 and 28 C, Fusarium solani f.
sp. phaseoli colonized between 90 and 100% of symptomatic

Figure 3-1. Conidiophores of Fusarium solani f. sp.
phaseoli observed on severely damaged roots of Vigna
radiata. Mature macroconidia (ma) were produced from
conidiophores on roots grown at 20-28 C, but not on roots
grown at 30-36 C. A,B, Branched monophialides (bm) were
observed emerging from the root epidermis (ep), from root
tips (rt), and from cortical cavities (cc) formed on
severely damaged roots when longitudinal splits developed in
the rot epidermis. The lower left-hand corner of Figure
3-1A indicates a cortical cavity on the root of V. radiata.
C, Unbranched monophialides (urn) developed from rhizospere
hyphae (rh). D, Sporodochia (sp) developed from hyphae
emerging from the root epidermis.

63

Figure 3-2. Deformed conidiophores of Fusarium solani f.
sp. phaseoli on roots of Viqna radiata grown at 20 C and pH
4.0. A, Conidiophores (co) formed branched complexes (be)
similar to branched monophialides at other hydrogen ion
concentrations, but mature macroconidia were not produced.
B, Deformed conidiophores were observed within cortical
cavities (cc) formed on severely damaged roots when
longitudinal splits developed in the root epidermis (ep). C,
Mycelia also remained disorganized in cortical cavities and
did not develop rudimentary conidiophores.

65

66
roots and between 21 and 83% of asymptomatic roots (Tables
3-5, 3-6, 3-7, and 3-8). The fungus was not isolated from
asymptomatic roots at 36 C, but it was isolated from
asymptomatic roots at all other temperatures. The decrease
in the percentage of asymptomatic roots colonized by F.
solani f. sp. phaseoli at 24-25 C as compared to 20 C was
generally significant for three experiments (P < 0.05)
(Tables 3-5, 3-7, and 3-8), but was not significant for the
experiment on the effects of [H+] on disease (Table 3-6).
An effect of [H+] on disease was observed at 25 C, but
not at 20 C (Table 3-2). At 20 C, disease was similar among
all [H+] tested and was consistent with other experiments in
which treatments were maintained at 20 C. At 25 C, fresh
weights of noninoculated and inoculated plants were not
different at pH 4.0 (P > 0.10), but they were different at
pH 5.0, 6.0, and 7.0 (P < 0.05). Fresh weights of
inoculated plants at 25 C and pH 4.0 were larger than fresh
weights of inoculated plants at 25 C and pH 7.0. Root
symptoms were dramatically reduced on inoculated plants
incubated at 25 C and pH 4.0, compared to root symptoms on
plants incubated at 25 C and pH 5.0, 6.0, and 7.0. The
individual effects of temperature and [H+] were best fit by
linear polynomial equations (P < 0.05). An interactive
effect between temperature and [H+] was not observed
(P > 0.10).
Numbers of propagules (colony-forming units/ml) of the
pathogen detected in nutrient solution at time zero

67
Table 3-5. Effects of temperature on the colonization of
roots of Viana radiata by Fusarium solani f. sp. phaseoli.
Temperature
(C)
Percent of colonized
root segments
Symptomatic
roots
Asymptomatic
roots
20
100 a^
59 b
24
100 a
21 c
28
94 a
49 b
32
z
38 be
36
—
0 d
Â¥ Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of F. solani f. sp.
phaseoli. Table values represent the means of three
replicates per treatment (one replicate per treatment for
each of three experiments). Data were transformed to the
arcsines of square roots; table values are presented as
detransformed numbers. Data from asymptomatic roots were
subjected to an orthogonal polynomial contrast analysis
and least-squares mean separation tests. Comparisons
between treatments equal to 100% and treatments < 100%
were conducted using paired t-tests for comparing
treatment means to a standard value; significant test
statistics were based on the standard errors of the means
of the normally distributed populations. Treatments
followed by the same letter were not different (P > 0.05).
z Symptomatic roots were not observed at 32 and 36 C.

68
Table 3-6. Effects of hydrogen ion concentration and
temperature on the colonization of roots of Vicrna radiata by
Fusarium solani f. sp. phaseoli.
PH
Temperature
(C)
Percent of colonized root segments
Symptomatic
roots
Asymptomatic
roots
4.0
20
92 az
63 b
25
100 a
75 b
5.0
20
100 a
46 b
25
100 a
36 b
6.0
20
98 a
63 b
25
100 a
47 b
7.0
20
100 a
72 b
25
100 a
44 b
z Twenty-
â– four, 1-cm-long
segments, for both
symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of F. solani f. sp.
phaseoli. Table values represent the means of three
replicates per treatment (one replicate per treatment for
each of three experiments). Data were transformed to the
arcsines of square roots; table values are presented as
detransformed numbers. Data from asymptomatic roots were
subjected to an orthogonal polynomial contrast analysis
and least-squares mean separation tests. Comparisons
between treatments equal to 100% and treatments < 100%
were conducted using paired t-tests for comparing
treatment means to a standard value; significant test
statistics were based on the standard errors of the means
of the normally distributed populations. Treatments
followed by the same letter were not different (P > 0.05).

69
Table 3-7. Effects of humidity and temperature on the
colonization of roots of Viqna radiata by Fusarium solani f.
sp. phaseoli.
Humidity
(mm Hg, VPD)X
Temperature
Percent of colonized
segments
root
(C)
Symptomatic
roots
Asymptomatic
roots
o
•
VO
20
98 a^
60
be
25
90 a
35
d
30
z
60
be
12.0
20
94 a
60
be
25
90 a
46
cd
30
—
70
b
x Humidity was measured as vapor pressure deficit (VPD) in
mm Hg; vapor pressure deficits of 6.0 and 12.0 mm Hg, at
25 C, correspond to 75 and 45% relative humidities,
respectively.
y Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of F. solani f. sp.
phaseoli. Table values represent the means of two
replicates per treatment (one replicate per treatment for
each of two experiments). Data were transformed to the
arcsines of square roots and then subjected to an
orthogonal polynomial contrast analysis and least-squares
mean separation tests; table values are presented as
detransformed numbers. Treatments followed by the same
letter were not different (P > 0.05).
z Symptomatic roots were not observed at 30 C.

70
Table 3-8. Effects of light quality and temperature on the
colonization of roots of Viqna radiata by Fusarium solani f.
sp. phaseoli.
Lamp-type
Temperature
Percent of colonized
segments
root
(C)
Symptomatic
roots
Asymptomatic
roots
Fluorescent
20
96 az
65
ab
25
95 a
42
b
Metal halide
20
96 a
84
a
25
97 a
32
c
High pressure
20
93 a
66
a
sodium
25
97 a
36
c
z Twenty-four, 1-cm-long segments, for both symptomatic and
asymptomatic roots within each treatment and experiment,
were sampled for the presence of F. solani f. sp.
phaseoli. Table values represent the means of three
replicates per treatment (one replicate per treatment for
each of three experiments). Data were transformed to the
arcsines of square roots and then subjected to an Analysis
of Variance procedure; table values are presented as
detransformed numbers. Treatments followed by the same
letter were not different based on least-squares mean
separation tests (P > 0.05).

71
increased as temperature increased during experiments on
temperature, [H+], and humidity (P < 0.05) (Tables 3-9, 3-
10, and 3-11), but not light quality (Table 3-12). The
relationships between temperature and the numbers of
propagules detected were best described by linear polynomial
equations (P < 0.03) (Tables 3-9 and 3-11). After 24 hr,
the numbers of propagules detected in nutrient solution
decreased dramatically in all experiments and remained low
for 1-10 days after inoculation in most experiments (Tables
3-9, 3-10, and 3-12). Numbers of propagules detected in
nutrient solution generally increased between 10 and 14 days
at 20 C (Tables 3-9, 3-10, and 3-12), except when the [H+]
was maintained at pH 4.0 and in the experiments with
humidity (Tables 3-10 and 3-11).
Species of Acremonium. Aspergillus. Cephalosporium.
Chaetomium. Cladosporium. Curvularia. Cvlindrocarpon.
Fusarium. Mucor. Penicillium. Stachvbotrvs. and Trichoderma
were isolated concommittantly with F. solani f. sp. phaseoli
from both inoculated and noninoculated treatments. Fusarium
spp., other than F. solani f. sp. phaseoli. isolated from
nutrient solution were identified to species based on their
growth on carnation leaf agar using the keys of Nelson
et al. (160); all isolates were keyed to Fusarium oxvsporum.
Species of Aspergillus. Chaetomium. Cladosporium. and
Penicillium were isolated most frequently; the numbers of
propagules detected in nutrient solution were generally
between 5 and 50 colony-forming units (cfu)/ml.

Table 3-9. Effects of temperature on the numbers of propagules of Fusarium solani f. sp.
phaseoli detected in nutrient solution over time during experiments on disease development
in Viana radiata.
Temperature
(C)
No. of
propagules
(cfu/ml)/day
0
1
5
10
14
20
409
A, abz
37
BC, a
19 C, a
9
C, a
56
B, a
24
368
A, a
7
B, a
3 B, a
3
B, a
7
B, b
28
436
A, ab
29
B, a
7 B, a
1
B, a
15
B, b
32
457
A, ab
33
B, a
16 BC,
a 11
C, a
14
BC, b
36
519
A, b
7
B, a
2 BC,
a 1
C,a
1
C, b
2 Table values represent the means of six replicates per treatment (two replicates per
treatment in each of three experiments). Data were transformed to square roots and then
subjected to an orthogonal polynomial contrast analysis when testing the effect of
temperature within each day and an Analysis of Variance procedure when testing the
effect of day within each temperature; table values are presented as detransformed
numbers. Treatments in rows followed by the same capital letter, and treatments in
columns followed by the same small letter, were not different based on least-squares
mean separation tests (P > 0.05).
to

Table 3-10. Effects of hydrogen ion concentration and temperature on the numbers of
propagules of Fusarium solani f. sp. phaseoli detected in nutrient solution over time
during experiments on disease development in Viana radiata.
pH
Temperature
(C)
No. of
propagules
(cfu/ml)/day
0
1
5
10
14
4.0
20
305
A, az
4
B, a
1
B, a
1
B, a
2
B, a
25
425
A, c
23
B, b
3
C, ab
4
C, a
1
C, a
5.0
20
319
A, a
4
C, a
2
C, a
22
C, b
74
B, be
25
394
A, be
12
B, b
3
C, b
2
C, a
4
C, a
6.0
20
351
A, ab
6
C, ab
3
C, a
13
C, b
107
B, be
25
379
A, b
15
B, b
5
BC,bc 2
C, a
10
BC, a
7.0
20
313
A, a
4
C, a
6
C, c
16
C, b
46
B, b
25
369
A, b
12
B, b
3
C, ab
2
C, a
6
C, a
z Table values represent the means of six replicates per treatment (two replicates per
treatment in each of three experiments). Data were transformed to square roots and then
subjected to an orthogonal polynomial contrast analysis when testing the effects of
hydrogen ion concentration and temperature within each day and an Analysis of Variance
procedure when testing the effect of day within each hydrogen ion concentration and
temperature combination; table values are presented as detransformed numbers.
Treatments in rows followed by the same capital letter, and treatments in columns
followed by the same small letter, were not different based on least-squares mean
separation tests (P > 0.05).
U>

Table 3-11. Effects of humidity and temperature on the numbers of propagules Fusarium
solani f. sp. phaseoli detected in nutrient solution over time during experiments on
disease development in Viqna radiata.
Humidity
(mm Hg, VPD)Â¥
Temperature
(C)
No. of
propagules
(cfu/ml)/day
0
1
5
10
14
6.0
20
426
A, az
1
B, a
9
B, a
121 B,a
111
B, a
25
476
A, b
6
B, a
3
BC, a
1 C, a
2
BC, a
30
527
A, c
32
B, b
8
CD, a
5 CD, a
16
BD, a
12.0
20
415
A, a
1
B,a
4
B, a
44 B,a
69
B, a
25
480
A, b
11
B, b
3
BC, a
1 C, a
1
BC, a
30
525
A, c
26
B, c
10
BD, a
2 CD, a
8
CD, a
y Humidity was measured as the vapor pressure deficit (VPD) in mm Hg; vapor pressure
deficits of 6.0 and 12.0 mm Hg, at 25 C, correspond to 75 and 45% relative humidities,
respectively.
z Table values represent the means of four replicates per treatment (two replicates per
treatment in each of two experiments). Data were transformed to square roots and then
subjected to an orthogonal polynomial contrast analysis when testing the effects of
humidity and temperature within each day and an Analysis of Variance procedure when
testing the effect of day within each humidity and temperature combination; table values
are presented as detransformed numbers. Data were highly variable between experiments
at days 10 and 14. Treatments in rows followed by the same capital letter, and
treatments in columns followed by the same small letter, were not different based on
least-squares mean separation tests (P > 0.05).
4*.

Table 3-12. Effects of light quality and temperature on the numbers of propagules of
Fusarium solani f. sp. phaseoli detected in nutrient solution over time during experiments
on disease development in Viana radiata.
Lamp type
Temperature
(C)
No. of propagules
(cfu/ml)/day
0
1
5
10
14
Fluorescent
20
404
A, a2
4
CD, a
2
C, a
14 CD,a
46
B, a
25
441
A, a
5
BC, a
4
BC, a
1 CD, b
8
B, b
Metal halide
20
434
A, a
3
C, a
2
C, a
11 C, a
45
B, a
25
443
A, a
5
B, a
3
B, a
1 B, b
4
B, b
High pressure
20
425
A, a
4
CD, a
2
D, a
13 CD,a
33
B, a
sodium
25
443
A, a
4
B, a
3
B, a
2 B, b
6
B, b
2 Table values represent
the means of
six :
replicates
per treatment (two replicates
i per
treatment in each of three experiments). Data were transformed to square roots and then
analyzed using Analysis of Variance; table values are presented as detransformed
numbers. Separate analyses were required to test for lamp and temperature effects within
each day and to test for an effect of day within each lamp and temperature combination.
Treatments in rows followed by the same capital letter, and treatments in columns
followed by the same small letter, were not different based on least-squares mean
separation tests (P > 0.05).

76
Periodically the numbers of propagules of Cladosporium spp.
detected in nutrient solution exceeded 200 cfu/ml. Fungal
contaminants were not correlated with any specific
treatment, nor were fungi correlated to anomalous root
symptoms on noninoculated plants. Carbon-based materials in
contact with the nutrient solution were found to be the
primary source of fungal contamination. Plastic tubing,
foam insulation, and black conduit tape were readily
colonized by fungi. Plastic tubing was periodically
replaced to reduce fungal contamination in the nutrient
solution. Species of Cladosporium. Chaetomium. and
Trichoderma were isolated most frequently from plastic
tubing and foam insulation; Cladosporium spp. were the
predominant fungi isolated from black conduit tape placed on
the upper surface of the 4-L tanks to reduce light
penetration into the hydroponic solutions.
Root length of L. sativa, cv 'Grand Rapids,' was not
significantly affected in bioassays by temperature, [H+],
humidity, and light quality experiments. Bioassays were
generally consistent between treatments within each
experiment, but differed between experiments. In the
experiments on the effects of temperature on disease, root
length of L. sativa varied between 23.4 and 27.3 mm after 4
days of incubation. In other experiments bioassays were
incubated for 3 days and root length was shorter, but root
length did not differ between treatments within each
experiment. Root length varied between 18.7 and 21.3 mm for

77
the [H+] and light quality experiments, and between 12.9 and
17.0 mm for the humidity experiment.
Temperature generally imparted the greatest affect on
element composition in foliar tissues of noninoculated
plants of V. radiata (Tables 3-13, 3-14, 3-15, and 3-16).
Boron and iron were positively correlated to increasing
temperature and were best described by linear polynomial
equations (P < 0.01) (Tables 3-13 and 3-15). Molybdenum was
negatively correlated to increasing temperature and was best
described by a linear polynomial equation (P < 0.01).
Calcium, potassium, and silicon were best described by
positive quadratic polynomial equations (P < 0.01), with
maximum concentrations observed between 24 and 32 C.
Copper, magnesium, and zinc were best described by negative
quadratic polynomial equations (P < 0.03), with minimum
concentrations observed between 24 and 32 C. The most
complex response to temperature was observed for Mn, which
increased in concentration between 20 and 24 C, decreased
between 24 and 28 C, and then increased between 28 and 36 C;
the response was best described by a cubic polynomial
equation (P < 0.01). No effect of temperature was observed
for either Na or P in noninoculated plants (P > 0.10).
Differences in element concentration between
noninoculated and inoculated plants were greatest at 20 C
for Ca, Cu, Fe, K, Mn, Na, P, Si, and Zn (P < 0.05) (Tables
3-13, 3-14, 3-15, and 3-16). Differences in element

Table 3-13. Effects of temperature on the elemental composition of foliage of Viana
radiata in experiments on disease development by Fusarium solani f. sp. phaseoli.
Element
Element composition
(mg-kg 1 or
%) of plants grown
at temperatures (C)
CON^
20
24
28
INOC
CON
INOC
CON
INOC
P
5910z
3580*
5413
4196*
5059
5109
Na
96
61*
111
76*
110
78*
K
10.2%
5.1%*
10.6%
8.6%*
11.5%
11.2%
Ca
1.8%
1.3%*
2.0%
1.9%
2.0%
1.9%
Mg
4720
4520
4234
4364
4075
4333
Zn
50
20*
39
27
32
28
Cu
8
2*
5
4
7
4*
Fe
71
63
77
74
102
91
Mn
103
42*
110
113
93
97
B
36
34
39
40
43
44
Mo
4
2*
2
2
1
1
Si
945
540*
1142
1093
1119
1111
y CON = noninoculated control plants; INOC = inoculated plants.
z Replicate tissue samples from each treatment were combined and processed as one bulked
sample per experiment. Table values represent the means of three bulked samples.
Significant differences, based on least-squares mean separation tests, in the
comparisons between noninoculated and inoculated plants at each temperature are
indicated by an asterisk (*) (P < 0.05).
00

Table 3-13. Continued.
Element
Element
composition (mg-kg-1 or %)
of plants grown
at
temperatures (C)
CON
32
INOC
CON
36
INOC
P
4632
4686
4348
4472
Na
92
88
102
96
K
10.3%
8.6%
9.5%
10.6%
Ca
2.0%
2.0%
1.7%
1.9%
Mg
3866
3904
4364
4558
Zn
34
35
44
50
Cu
7
7
13
14
Fe
97
89
102
100
Mn
109
109
121
139
B
57
61
70
76
Mo
1
1
1
1
Si
1291
1351
748
793

Table 3-14. Effects of hydrogen ion concentration and temperature on elemental
composition of foliage of Viana radiata in experiments on disease development by Fusarium
solani f. sp. phaseoli.
Element
Element composition
(mg•kg 1 or
%) of plants grown at 20 C and pH
CON^
4.0
INOC
CON
5.0
INOC
6
CON
. 0
INOC
7
CON
.0
INOC
P
5105z
2886*
4881
2819*
4772
2882*
4681
3002*
Na
66
49
80
40*
65
37
53
46
K
8.9%
4.1%*
8.6%
3.6%*
8.1%
3.6%*
7.4%
4.2%*
Ca
1.7%
1.0%*
1.6%
0.9%*
1.7%
1.0%*
1.7%
1.0%*
Mg
3985
2996
3955
3587
4328
3591
4542
4142
Zn
80
35*
66
38*
55
28*
33
30
Cu
14
7*
10
7*
8
6
3
5
Fe
182
98*
153
81
121
77
101
80
Mn
124
39*
99
31*
106
43*
41
23
B
34
30
33
31
34
31
36
32*
Mo
3
1*
3
1*
3
2*
3
2*
Si
667
364*
673
266*
731
354*
834
326*
y CON = noninoculated control plants; INOC = inoculated plants.
z Replicate tissue samples from each treatment were combined and processed as one bulked
sample per experiment. Table values represent the means of three bulked samples.
Significant differences, based on least-squares mean separation tests, in the
comparisons between noninoculated and inoculated plants at each hydrogen ion
concentration and temperature are indicated by an asterisk (*) (P < 0.05).
00
o

Table 3-14. Continued
Element
Element composition
(mg-kg-1 or
%) of plants grown
at 25 C and pH
CON
4.0
INOC
CON
5.0
INOC
6
CON
. 0
INOC
7
CON
.0
INOC
P
5391
4442*
4482
3910
4541
3679*
4530
3881
Na
154
94*
143
73*
171
76*
167
47*
K
6.9%
6.9%
6.5%
6.3%
6.2%
6.2%
6.7%
5.1%
Ca
2.1%
2.1%
2.1%
2.0%
2.0%
2.0%
2.1%
2.0%
Mg
3490
3762
3492
3818
3860
3956
4211
4546
Zn
77
66
45
39
37
30
26
21
Cu
14
12
8
9
5
4
3
1
Fe
331
146*
116
128
90
85
88
87
Mn
181
90*
101
108
88
111
41
57
B
34
31
33
33
34
36
35
36
Mo
1
1*
1
1
1
1
1
1
Si
1158
1135
1166
1222
1085
1216
1300
1428

Table 3-15. Effects of humidity and temperature on the elemental composition of foliage
of Vigna radiata in experiments on disease development by Fusarium solani f. sp. phaseoli.
Element
Element
composition
(mg•kg-1
or %) of plants grown
temperature (C)x
i at 6.0 mm
Hg, VPD and
20
25
30
CON^
INOC
CON
INOC
CON
INOC
P
5572 z
3240*
5052
3923*
4330
4225
Na
84
49
116
91
201
128*
K
11.6%
3.8%*
9
. 0%
6.3%*
7.3%
7.0%
Ca
1.9%
1.2%*
2
.2%
1.8%*
1.9%
1.8%
Mg
4480
3963
4238
4316
3630
3695
Zn
40
17*
37
24*
27
26
Cu
10
8
10
8
9
11
Fe
99
69*
100
89
110
107
Mn
93
52*
84
91
83
88
B
34
30
38
29
41
41
Mo
3
2*
1
1
1
1
Si
819
516
1059
950
775
768
x Humidity
was measured
as vapor pressure <
deficit
(VPD); 6.0 mm
Hg VPD and
12.0 mm Hg VPD
correspond to 70% and 50% relative humidity, respectively, at 25 C.
y CON = noninoculated control plants; INOC = inoculated plants.
z Replicate tissue samples from each treatment were combined and processed as one bulked
sample per experiment. Table values represent the means of two bulked samples.
Significant differences, based on least-squares mean separation tests, in the
comparisons between noninoculated and inoculated plants at each humidity and temperature
are indicated by an asterisk (*) (P < 0.05).
oo
NO

Table 3-15. Continued.
Element
Element
composition
(mg-kg 1 or %) of plants grown
temperature (C)x
at 12.0 mm
Hg, VPD at
CON
20
INOC
CON
25
INOC
CON
30
INOC
P
5080
3356*
4682
4653
5102
4942
Na
80
52
88
100
198
142*
K
10.0%
4.9%*
8.5%
7.6%
9.8%
10.5%
Ca
1.9%
1.2%*
2.0%
1.8%
2.0%
1.7%
Mg
4473
4150
4358
4363
3873
3977
Zn
36
21*
32
25
34
29
Cu
6
2*
4
3
8
7
Fe
92
76
96
88
110
101
Mn
94
58*
99
86
92
88
B
39
36
43
39
47
46
Mo
4
2*
1
1
1
1
Si
824
461
1080
921
767
755
00
OJ

Table 3-16. Effects of light quality and temperature on the elemental composition of
foliage of Viana radiata in experiments on disease development of Fusarium solani f. sp.
phaseoli.
Element
Element composition (mg-kg-1 or
%) of plants grown
under lamp
-type
High
pressure
sodium
Metal
halide
Temperature
(C)
Temperature (C)
20
25
20
25
C0Nx
INOC
CON
INOC
CON
INOC
CON
INOC
P
4228^
3166*
4102
3469*
4943
3377*
4559
3902*
Na
58
46
166
81*
71
53
174
135
K
5.6%
3.2%*
5.7%
5.4%
7.1%
3.6%*
6.8%
5.8%
Ca
1.6%
1.2%*
2.0%
1.9%
1.6%
1.2%*
12.0%
1.8%*
Mg
4148
3998
3529
3794
4439
4211
3748
4193
Zn
32
17*
25
17
59
37*
56
51
Cuz
81
82
74
72
70
87
76
84
Fe
87
75
92
82
55
45
56
56
Mn
94
80*
86
100
108
77*
105
147*
B
33
33
43
41
36
37
42
57
Mo
2
1*
<1
<1
2
<1*
<1
<1
Si
1021
768*
1388
1378
767
529*
1039
1095
x CON = noninoculated control plants; INOC = inoculated plants.
y Replicate tissue samples from each treatment were combined and processed as one bulked
sample per experiment. Table values represent the means of three bulked samples.
00

Table 3-16. Continued.
Element
Element
composition (mg-kg 1 or %) of
plants grown
under
lamp-type
Fluorescent
Temperature
(C)
20
25
CON
INOC
CON
INOC
P
4550
3495*
4400
3648*
Na
59
57
167
92*
K
5.8%
4.6%
6.9%
5.7%
Ca
1.8%
1.3%*
2.2%
1.9%*
Mg
4244
4036
3918
4216
Zn
37
27
29
24
Cu
79
74
65
65
Fe
83
71*
91
82
Mn
91
66
101
116
B
35
34
44
45
Mo
2
1*
1
1
Si
1025
749*
1452
1342
Significant differences, based on least-squares mean separation tests, in the
comparisons between noninoculated and inoculated plants under each lamp type and at each
temperature are indicated by an asterisk (*) (P < 0.05).
z Copper contamination from centrifugal aspirator assemblies of humidifiers inflated the
concentration of copper in foliar tissues.
CO

86
concentration between noninoculated and inoculated plants
were not observed for B and Mg for any temperature
(P > 0.10), except for B at 20 C and pH 7.0 (Table 3-14).
Differences in element concentration between noninoculated
and inoculated plants generally decreased as temperature
increased in all experiments (Tables 3-13, 3-14, 3-15, and
3-16); element concentration of tissues from noninoculated
and inoculated plants were similar at 28-36 C (P > 0.10)
(Table 3-13).
Effects of [H+] on element concentration were not
observed for Ca, Mo, P, Si, Na, and Mg (P > 0.05) (Table
3-14). Boron was positively correlated with increasing pH
and its response was best described by a linear polynomial
equation (P = 0.019). Copper, iron, potassium, manganese,
and zinc decreased with increasing pH at both 20 and 25 C
and the responses were best described by linear polynomial
equations (P < 0.05). Differences in element concentration
between noninoculated and inoculated plants were greatest at
20 C and appeared to be little affected by [H+]
(Table 3-14).
Effects of humidity on element concentration in either
noninoculated or inoculated plants were not observed for any
elements tested. Differences between noninoculated and
inoculated plants were generally not affected by humidity
(Table 3-15) nor by light quality (Table 3-16). Effects of
light quality on element concentration were not observed for
Ca, Cu, K, and Na (P > 0.10) (Table 3-16). Iron and silicon

87
were suppressed, and Mn, P, and Zn increased, in plants
grown under MH lamps (P < 0.05). Boron and magnesium were
slightly lower in plants grown under HPS lamps compared to
FL and MH lamps (P < 0.05). The accumulation of Mo was
slightly increased in plants grown under FL lamps compared
to MH and FL lamps (P < 0.05).
Discussion
Microbial contamination of space-based CELSS will
likely include fungi that are ecologically adapted to
survival on plants originally grown in terrestrial
environments. For example, 29 of 57 genera of fungi
isolated during the Apollo 14 and 15 missions (218) were
found in the United States Department of Agriculture (USDA)
host index of plant diseases (150). It is difficult to
ascertain if microbial contamination of CELSS modules will
constitute a realistic threat to plant health. Microgravity
may alter many aspects of plant-microbe interactions such
that new and unique pathological relationships develop.
Although, the physiologies of plants (46,87,121,155,156,202)
and microbes (2,170,214,216,240) are altered by
microgravity, no studies on plant-microbe interactions in
the space environment have been reported.
As stated earlier, hydroponic plant production systems
will be utilized in space-based CELSS (30,101,124,166,219),
and it seems apparent that a clear understanding of disease
etiology, epidemiology, and control in terrestrial

88
hydroponic systems will be essential in developing an IPM
program for CELSS. However, some controversy exists on the
importance of root diseases in hydroponic systems in
terrestrial environments.
The significance of root diseases in hydroponic systems
has been questioned by several researchers (51,72,100,
177,212). For example, Funck-Jensen and Hockenhull (72)
reported that lettuce grown under good conditions in a
nutrient film technique (NFT) system appeared to be highly
resistant to attack by Pvthium spp. (72) . Hockenhull and
Funck-Jensen (100) reported that low disease in a
recirculating NFT system may have been due to the dilution
of root exudates that subsequently reduced the inoculum
potential of the pathogen. Furthermore, Rattink (184)
suggested that the physical attributes of the ebb and flow
hydroponic system altered dispersal of infective propagules,
which subsequently reduced the severity of disease.
Other researchers have indicated that root disease in
hydroponic systems can be rapid and severe on tomato
(45,145,225), carnation (182), cucumber (165,211), lettuce
(207,208), spinach (15,77,210), and bean (4,84). In
addition, hydroponic systems have been used effectively to
screen plant culivars for resistance to root pathogens
(5,6,23,239). Guerra and Anderson (84) suggested that
hydroponic culture may predispose bean plants to infection
by F. solani f. sp. phaseoli. Rattink (182) reported that
the spread of disease caused by F. oxvsporum f. sp. dianthi

89
(Prill. & Del.) Snyd. & Hans, on carnations was very rapid
in both rockwool and NFT systems.
In the current study, the severity of disease caused by
F. solani f. sp. phaseoli in V. radiata and the production
of secondary inoculum were very sensitive to temperature,
and to a lesser extent, [H+], but neither was affected by
humidity and light quality. Disease was greatest at 20 C
and declined steadily until at 30 C plant weight differences
were not observed between noninoculated and inoculated
plants. The effects of temperature on the severity of
disease were similar to those on other bean cultivars
infected with F. solani f. sp. phaseoli (33,146,147) and F.
oxvsporum f. sp. phaseoli Kendr. & Snyd. (186).
Stanghellini and coworkers (15,77,207) have described
several pathosystems in hydroponic systems that are
sensitive to temperature and have suggested that the
manipulation of temperature might be used as a cultural
control method for certain root pathogens in hydroponic
systems (15).
The numbers of propagules of F. solani f. sp. phaseoli
in nutrient solution were monitored over time in several
experiments. The numbers of propagules recovered at time
zero generally increased as temperature increased. The
numbers of propagules recovered at time zero represented the
differences among treatments at 20-40 min after inoculation,
which was the actual time after inoculation before samples
could be obtained. Differences among treatments at time

90
zero were believed to be due to the effects of temperature
on spore attachment. In a separate study, the numbers of
macroconidia of F. solani f. sp. phaseoli attached to roots
of V. radiata decreased dramatically when plants were
inoculated at temperatures greater than 30 C (Chapter 4).
Therefore, the numbers of propagules recovered from nutrient
soultion at time zero in the present study may have been
greater at higher temperatures because they were unable to
attach to root surfaces. In addition, the lack of recovery
of F. solani f. sp. phaseoli from asymptomatic roots at 36 C
(Table 3-5) was probably due to the effects of high
temperature on spore attachment and fungal growth. Spore
attachment and fungal growth have been shown to be
dramatically reduced, or prevented, at 35 C (Chapter 4).
Alteration of [H+] has received very little attention
as a possible control method for root disease in hydroponic
systems. In the current study, [H+] was found to alter both
the severity of disease and the production of secondary
inoculum by F. solani f. sp. phaseoli. At 20 C, disease was
not altered by changes in [H+]; however, secondary inoculum
was not produced at pH 4.0, but was produced at pH 5.0, 6.0,
and 7.0. At 25 C, fresh weights of inoculated and
noninoculated plants were not different when plants were
maintained at pH 4.0. In comparison, plant fresh weights of
inoculated plants were 20-27% less than those of
noninoculated plants when plants were maintained at pH 5.0,
6.0, and 7.0. Thus, a [H+] near pH 4.0 can reduce the

91
severity of disease and reduce secondary inoculum
production, but the effects are dependent on temperature and
on the timing of application of the [H+] treatment
(Chapter 4).
In other studies with F. solani f. sp. phaseoli. the
severity of disease was greater when pH was maintained at
4.0, compared to pH 5.0-7.0, during inoculation; the
severity of disease was very low on plants inoculated at
pH 7.0 (Chapter 4). Temperature and [H+] were shown to
greatly affect attachment of macroconidia to roots of V.
radiata in nutrient solution (Chapter 4); the optimum for
macroconidium attachment was at 20 C and pH 4.0. In the
current experiments, the pH was maintained at 6.0 for all
treatments during the first 24 hr after inoculation, and
then adjusted to pH 4.0, 5.0, 6.0, and 7.0. The procedure
was reversed for studies on the effects of [H+] on spore
attachment (Chapter 4); treatments were adjusted to pH 4.0,
5.0, 6.0, and 7.0 prior to inoculation, maintained for
24 hr, and then adjusted to pH 6.0. The effects of [H+] on
the severity of disease caused by F. solani f. sp. phaseoli
in V. radiata appear to be governed by two separate
mechanisms. One mechanism appears to affect spore
attachment to roots and a second affects post-attachment
processes on or in the plant host. If confirmed, each
mechanism might be used independently to control different
aspects of the disease cycle.

92
A few studies (19,72,236) have been conducted on the
effects of [H+] on disease in hydroponic systems, but they
are not in agreement. Funck-Jensen and Hockenhull (72)
found that [H+], in the range of pH 4.2-7.8, had no effect
on disease caused by a Pvthium sp. on lettuce in a
hydroponic system. Conversely, Bingham and Zentmyer (19)
observed less severe disease caused by Phvtophthora
cinnamomi Rands on avocado and Yoganathan et al. (236)
observed more severe disease caused by Pvthium debarvanum
Hesse on lettuce when pH was maintained at or below 4.5, as
compared to when pH was maintained at or above 6.0. The
current study is the first to investigate the effects of
[H+] on a disease caused by a Fusarium sp. in a hydroponic
nutrient solution. Obviously, additional research is re¬
quired with other pathosystems to determine if [H+] affects
disease in other hydroponic systems in a similar manner.
One limitation to understanding the role [H+] has in
disease in hydroponic systems has been inadequate
presentation of [H+] used during experimental procedures.
The pH of the nutrient solution has been reported in several
studies in which hydroponic systems were used to conduct
root pathological research (66,84,117,176,177), but it has
not been reported in many other studies involving root
pathogens in hydroponic systems (5,15,45,51,77,
95,100,106,145,182,183,184,207,208,212,225,226). Slow
disease progress reported in some studies (51,100,184,212)
may not be due to inherent qualities of the hydroponic

93
systems, as some have suggested (100,177,184), but actually
due to a bit of serendipity. Hoagland and Arnon (99), in
one of the most frequently cited papers for identifying the
composition of the nutrient solution in hydroponic systems,
indicated that [H+] can be regulated within the range of pH
5.0-6.5. However, in studies in which the pH was reported
(66,84,117,176,177) the [H+] was maintained between pH 6.0
and 6.7. Spore attachment to roots of V. radiata and the
severity of disease caused by F. solani f. sp. phaseoli have
been shown to be reduced between pH 6.0 and 7.0 (Chapter 4).
Based on the results from the current study, and from the
experiments in Chapter 4, it seems possible that low levels
of disease reported by some workers (51,100,184,212) may
have been due to maintaining the pH of the nutrient solution
above 6.0. Clearly, this possibility should be considered
in future studies. Nutrient solutions maintained at
pH 6.0-6.5 appear to have become the accepted standard for
plant pathological research in hydroponic systems. This
standard should be reevaluated for two reasons. First, [H+]
may be usable as a simple and low-cost method for managing
plant diseases in hydroponic systems. Secondly, an
understanding of the biology of root pathogens and their
roles in disease in hydroponic systems requires that the
effects of [H+] on root pathogens be determined.
One explanation for the reluctance to utilize [H+]
below pH 6.0 may be a perception that acidic conditions
cannot be readily tolerated by vegetable plant species in

94
hydroponic systems. Hydrogen ion concentrations below pH
5.0 may not be as detrimental to plant species grown in
hydroponic systems as they might be to plants grown in soil
systems. Aluminum toxicity is a major concern in acid soils
(138) and generally is due to the increasing availability of
the toxic Al3+ ion species below pH 5.0 (138). However,
nutrient solutions using high quality water and inorganic
salts should be relatively free of aluminum species,
provided that the physical hydroponic system does not
contain aluminum materials in direct contact with the
nutrient solution. Several plant species have been grown in
nutrient solutions in which pH was maintained at or below
4.5 without significant detrimental effects on plant growth
(19,68,189,227). Legume and cereal crops tested by
Findenegg (68) appeared to be less affected by [H+] near pH
4.0 than were members of other plant families. In contrast,
Tolley-Henry and Raper (68) reported significant reductions
in dry matter, NH4 + assimilation, and new leaf production
with soybeans grown at pH 4.1, compared to those grown at
pH 5.1 and 6.1. The effect was attributed to high acidity
impairing uptake of NH4+ by Glycine max (L.) Merr. (68).
Clearly, the effects of [H+] on the severity of disease in
plants grown in hydroponic systems have been inadequately
studied. Results presented here and in Chapter 4 support
the conclusion that greater attention should be paid to both
testing and reporting the effects of [H+] in root pathology
research conducted in hydroponic systems.

95
Not all components of the hydroponic system used for
these studies appeared to affect disease caused by F. solani
f. sp. phaseoli in V. radiata. Humidity appeared to have no
significant effect on inoculated and noninoculated plants at
20, 25, or 30 C. In addition, humidity appeared to be
ineffective in altering the production of secondary inoculum
on roots of V. radiata, and it had little influence on the
composition of elements in trifoliate leaves of V. radiata.
The three HID lamp treatments also had little effect on
the severity of disease, but some differences in element
composition of plants grown under separate lamp treatments
were observed. Spectral qualities of HID lamps have been
shown to affect element composition of plant foliage
(11,108) and plant growth (108), but the significance of
these changes in relation to plant disease under artificial
light is uncertain. It was hypothesized that the lack of
ultraviolet light and visible light between 400 and 480 nm,
which occurred with HPS lamps but not with MH and FL lamps
(Figure 3-3), might reduce plant resistance under HPS lamps.
Lamps low in ultraviolet light have been shown to induce
iron deficiency in some plants (26,27), and induced iron
deficiency has been shown to increase the severity of
disease caused by F. solani f. sp. phaseoli in P. vulgaris
(84). However, results from the current study do not
support this hypothesis. Further research is recommended
with other root pathogens to determine if spectral qualities

Figure 3-3. Spectral photon flux (/nmol-s 1 • in-2) for high
pressure sodium, metal halide, and fluorescent lamps. Lamp-
to-plant distances were adjusted for each type of lamp to
provide a standard 300 /nmol • s-1 *m-2 of photosynthetically
active radiation between 400 and 700 nm. Infrared spectral
photon flux was greatest for high pressure sodium lamps,
where an emission peak was centered at 820 nm. Short-
wavelength emissions below 450 nm were lowest for high
pressure sodium lamps.

SPECTRAL PHOTON FLUX (/¿mol • s 1 • m 2 . nrn 1)
97
12.0
HIGH PRESSURE SODIUM
1 1
.
•r-
LJ
L_
10.0-- METAL HALIDE
B.0--
300 400 500 600 700 800 900
WAVELENGTH (nm)
1000 1100

98
of different HID lamps can alter disease in closed plant
growing systems.
Toxic microbial metabolites have been implicated in
root death in hydroponic systems (104) and as the cause of
reduced plant vigor in ground-based CELSS (76). Toxigenic
Fusarium spp. also have been shown to induce vascular
browning of tomato, cotton, cabbage, and pea plants
(52,234). A bioassay was developed to determine whether
plant-growth inhibiting or plant-growth promoting compounds
accumulated in nutrient solutions of various treatments.
Bioassay results were negative for all experiments; root
length of germinating L. sativa. cv. 'Grand Rapids,' did not
appear to be affected by treatment parameters within each
experiment. These results are consistent with a study by
Hurd (102), in which no detrimental effects on seed
germination and seedling growth were observed when testing
nutrient solution from hydroponic systems used to grow
tomato plants. However, only a few studies have
investigated the effects of nonpathogenic microorganisms on
plant health in hydroponic systems (6,174,199), and it might
be possible that the bioassay used in the current
experiments was not sensitive to plant-growth altering
compounds present in the nutrient solutions. Several
alternative plant bioassays for the detection of
fusariotoxins have been reviewed (107). Additional research
is required before conclusions can be made on the occurrence
of plant-growth altering compounds in hydroponic systems.

99
The role of plant nutrition in disease caused by F.
solani f. sp. phaseoli in V. radiata is also questionable.
In the present study, element composition of noninoculated
plants was altered by temperature, [H+], and light quality,
but not by humidity. Concentrations of P, Na, K, Ca, Zn,
Cu, Fe, Mn, Mo, and Si in trifoliate leaves of inoculated V.
radiata plants were dramatically reduced at 20 C, where the
severity of disease was greatest. In addition, as disease
was reduced by altering temperature or [H+], differences in
element concentrations between noninoculated and inoculated
plants decreased. The effects of [H+] on cation levels in
trifoliate leaves of V. radiata were consistent with the
effects of [H+] on cation availability in nutrient solutions
reported by Cline et al. (41). Although data support the
hypothesis that nutrient content of V. radiata leaves can be
affected by disease induced by F. solani f. sp. phaseoli. it
is unclear whether the reductions in specific elements
influenced plant fresh weight differences between
treatments. Element compositions of severely diseased
plants at 20 and 24 C were not below deficiency thresholds
reported for Viqna spp. and Phaseolus spp. (37,185). Berry
and Wallace (17) described the following three phases for
nutrient dose vs plant yield curves: a deficiency phase in
which yield increases as dose increases, a tolerance plateau
at which yield remains level as dose increases, and a
toxicity threshold beyond which yield decreases as dose
increases. It is likely that the high ionic strength of the

100
nutrient solution permitted adequate assimilation of
essential elements, even under conditions of severe root
disease. Thus, the critical levels for deficiencies (17)
probably were not exceeded and element concentrations in
trifoliate leaves remained on the tolerance plateaus.
Therefore, the current study does not conflict with the
report by Guerra and Anderson (84), which indicated that
extreme Fe and B deficiencies increased root disease caused
by F. solani f. sp. phaseoli in Phaseolus vulgaris L.
Results from the current study support the conclusion that
element composition of V. radiata can be altered by
environmental conditions and by root disease caused by F.
solani f. sp. phaseoli. but reductions in element
concentrations did not appear to contribute to plant fresh
weight differences observed between treatments.
Plant disease management is just one of several
daunting challenges to the design and operation of space-
based CELSS. It is likely that new concepts in plant
pathology will be required for managing plant diseases in
the unique environments found in space. Effects of gravity
and radiation have been neglected in terrestrial plant
pathology, but they cannot be ignored in space. Although
there is an extreme paucity of information in the nascent
discipline of CELSS pest management, the application of
information obtained from research into the biology,
etiology, and epidemiology of phytopathogens in terrestrial

101
hydroponic systems should be applicable to future space-
based CELSS.

CHAPTER 4
EFFECTS OF TEMPERATURE AND HYDROGEN ION CONCENTRATION ON
ATTACHMENT OF MACROCONIDIA OF FUSARIUM SOLANI F. SP.
PHASEOLI TO ROOTS OF VIGNA RADIATA IN HYDROPONIC
NUTRIENT SOLUTION
Introduction
Attachment of fungal structures to plant surfaces was
defined by Nicholson (161) to include all mechanisms, both
mechanical and chemical, through which fungi maintain
initial contact with the host tissue. The term adhesion was
employed to ". . .describe phenomena of attachment requiring
either the presence of adhesive materials or at least the
modification of the cuticle by the fungus. . ." (page 74,
cit. 161). In an ambient air environment, attachment of
fungal spores to plant surfaces may be a prerequisite for
pathogenesis (162). A conventional view of fungal
attachment to foliar surfaces includes entrapment of the
infective propagules on leaf projections followed by the
secretion of adhesive materials (161). The process of spore
attachment to plant roots grown in nutrient solution may be
conceptually similar to the process of spore attachment to
above-ground plant surfaces. Fluid dynamics of nutrient
solution and air flowing past plant surfaces should be
similar. The principles of laminar flow, turbulance, fluid¬
shearing forces, eddy formation, convective heating, and
boundary-layer dynamics described for air movement through
102

103
crop canopies (187) should be applicable to fluid movement
around root systems in liquid matrices. Dramatic
differences certainly exist between roots grown in nutrient
solution and plant surfaces in the ambient-air environment,
but spore attachment to root surfaces in nutrient solution
may also be a prerequisite for pathogenesis.
Macroconidia of Fusarium solani (Mart.) Appel. & Wr.
f. sp. phaseoli (Burk.) Snyd. & Hans, were observed to
attach directly to root surfaces of Vigna radiata (L.)
Wilczek in nutrient solution at various combinations of
temperature and hydrogen ion concentration ([H+]) (Chapter
3). Macroconidia attached via terminal and foot cells, and
appeared to attach in greater numbers to second-order roots
with root hairs than to other root orders (Chapter 3). In
addition, temperature and [H+] were found to have both
singular and interactive effects on disease caused by F.
solani f. sp. phaseoli in V. radiata. In a preliminary
experiment of the current study, low numbers of macroconidia
were observed on roots in temperature treatments that
coincided with low levels of disease in pathogenicity tests
reported in a separate study (Chapter 3).
Based on the results presented in Chapter 3, it was
hypothesized that temperature and [H+] can alter the
inoculum load on plant roots by affecting spore attachment,
and that these effects might correlate to differences in the
severity of disease among treatments. If substantiated,
temperature and [H+] might be used as effective methods in

104
the control of root diseases caused by Fusarium spp. in
hydroponic sytems. Thus, the objectives of this study were
1) to determine if macroconidia of F. solani f. sp. phaseoli
attached preferentially to different root orders of
V. radiata; 2) to evaluate the effects of temperature and
[H+] on spore attachment to, and germination on, roots of
V. radiata; and 3) to determine if different inoculum loads
on plant roots could influence different levels of plant
disease.
Materials and Methods
Inoculum of F. solani f. sp. phaseoli (isolate F-28A,
obtained from A. J. Anderson, Utah State University, Logan,
Utah) was prepared on modified carnation leaf agar (MCLA)
(Chapter 2). Four in vitro experiments were conducted to
determine the effects of temperature and [H+] on the radial
growth rate of fungal colonies and on the elongation of
macroconidium germ tubes. Tests were conducted on either
nutrient solution agar (NSA) (Chapter 2) or MCLA. When
temperature was the treatment variable, the pH was adjusted
to 6.0-6.5. When the pH was the treatment variable it was
adjusted between 3.0 and 11.0, while temperature was held
constant at 25 C. Chemicals were obtained from Sigma
Chemical Co. (St. Louis, MO, 63178). The [H+] was adjusted
using 0.01 N HNO3 and 0.02 N KOH. Radial growth rates of
F. solani f. sp. phaseoli were determined by placing one,
2.0-mm3 block of agar from a 14-day-old MCLA culture onto

105
each of 10 plates of fresh MCLA. Cultures were maintained
for 10 days in a Rheem 23L incubator (Rheem Manufacturing
Co., Asheville, NC, 28804) under a 12-hr diurnal period; a
photon flux of 70 /¿mol • s-1 «m-2 was maintained during the
light cycle. The following temperatures were tested: 15,
20, 22, 25, 27, 30, 32, and 35 C; the experiment was
conducted twice. The experiment on the effects of [H+] on
radial growth of F. solani f. sp. phaseoli was conducted
three times.
To determine the effects of temperature and [H+] on
germ tube elongation, macroconidia of Fusarium solani f. sp.
phaseoli were collected from 14-day-old MCLA cultures in
sterile deionized water (SDIW). Inoculum was adjusted to a
density of 4.0 x 104 macroconidia/ml. A hemacytometer was
used to quantify inoculum in this and all subsequent
experiments. Ten plates of NSA per treatment were
preincubated at specific temperatures. Ten, 1.0-/¿1 drops of
the macroconidium suspension were placed on the surface of
NSA. Cultures were incubated for 6 hr and then 7.0 ml of a
0.05% trypan blue stain (Chapter 2) were dispensed onto the
agar surface of each plate. Ninety germ tubes from each
treatment, per experiment, were measured using a Sony
DXC-3000 color video camera (Sony Corporation, Japan)
mounted on a Nikon Optiphot compound microscope (Nikon,
Inc., Norcross, GA, 30093). The video image was
electronically combined with the Microcomp® Integrated Video
Image Analysis System (Southern Micro Instruments, Inc.,

Atlanta, GA, 30339), as was described in Chapter 2. Each
experiment was conducted three times.
106
Statistical analyses were conducted with the
Statistical Analysis System (SAS) (SAS Institute, Inc.,
Cary, NC, 27512). Data from in vitro experiments were
analyzed by regression or orthogonal polynomial contrasts
(213). Coefficients for orthogonal polynomials were
estimated using the interactive matrix language (PROC IML)
program within SAS because treatments were unequally spaced.
During preliminary experiments on the in situ effects
of temperature and [H+] on spore attachment, macroconidia
were observed to attach differentially to distinct portions
of the roots of V. radiata. In order to quantify the
differences in spore attachment, 7- to 8-day-old plants of
V. radiata were produced in seed-germination trays
(Chapter 2). For each experiment, three plants were placed
into a 600-ml beaker containing 500 ml of nutrient solution
(Chapter 2). Macroconidia were added to 500 ml of fresh
nutrient solution in a second beaker and the density
adjusted to 4000 propagules/ml by determining the numbers of
spores in 10, 1.0-/xl samples transferred from the
macroconidium suspension to NSA. A third beaker contained
500 ml of fresh nutrient solution and was used to rinse
loosely attached macroconidia from roots following
inoculation. The temperature and pH of the nutrient
solution samples were maintained at 25 C and 5.0,
respectively. Temperature of the nutrient solution was

107
maintained using a Magni Whirl water bath (Blue-M Co., Blue
Island, IL, 60406). Compressed air was passed through a
0.45-jLtm, Gelman Mini-Capsule, cartridge filter (Gelman
Sciences, Inc., Ann Arbor, MI, 48106) and then bubbled
through the nutrient solution containing V. radiata
seedlings. Plants and inoculum were incubated for 1 hr
under six, 20-watt, fluorescent lamps which provided a light
intensity of 170-180 /¿mol* s-1*m“2 at the level of the plant
foliage. Inoculum was then transferred to a 600-ml beaker
wrapped with 4.0 mm of closed-celled foam tape (Macklenburg-
Duncan, Oklahoma City, OK, 73118). Plants were exposed to
inoculum individually for 5 min, and then rinsed for 30-40
sec. During inoculation macroconidium suspensions were
agitated gently using magnetic stirrers. Roots were stained
for 5 min in 0.05% trypan blue stain and placed in a
clearing solvent of lactic acid, glycerol, and water
(Chapter 2). Morphologically distinct root-types, termed
root orders in accordance with the morphometric method of
root classification described by Fitter (70), plus the lower
hypocotyls of each plant were dissected and mounted on
microscope slides. The numbers of macroconidia attached to
the lower hypocotyl, second-order roots with root hairs,
second-order roots without root hairs, first-order roots,
and root tips were estimated by counting macroconidia
present on 11 randomly selected root segments of each root
order. Observations were made at a magnification of 200x
using a Nikon Optiphot compound microscope; each field-of-

108
view was 0.9 mm in diameter. Therefore, the total sum of
macroconidia for each set of 11 randomly selected fields-of-
view, per root order, represented the numbers of
macroconidia present on one linear centimeter of root
tissue. The experiment was conducted five times.
Two different statistical analyses were utilized to
determine the quantitative differences in spore attachment.
In the first analysis unadjusted data representing the
numbers of macroconidia attached per linear cm of each root
order were transformed to logs and subjected to an Analysis
of Variance followed by Fischer's Least Significant
Difference test (P < 0.05). In the second analysis data
were adjusted to compensate for the increased area of root
hairs present on second-order roots and to compensate for
the different diameters of the root orders. The area of an
"average" root hair was calculated using the following
formula:
A = 2nr * L
where r was the radius and L the length of an average root
hair. To experimentally calculate r and L, 10 second-order
root segments possessing root hairs were mounted in water on
a microscope slide. The width and length of 150 root hairs
(15 per root segment) were measured using the video image
analysis system in an identical manner as was used to
measure the lengths of macroconidium germ tubes. The
numbers of root hairs per unit area of root surface were
estimated by photographing 15 different sections of second-

109
order roots with root hairs, at standardized angles and
magnifications, using a Hitachi S-530 scanning electron
microscope (Hitachi Instruments, Inc., Danbury, CT, 06810).
Root segments were prepared for scanning electron microscopy
(SEM) as outlined in Chapter 5. The area recorded in each
photograph was estimated as 0.26 mm2. The appropriate
mathematics were utilized to then calculate the increased
surface area created by root hairs on 1.0 mm2 of second-
order roots. The area of root and hypocotyl segments were
calculated in a similar manner. Adjusted data were analyzed
in an identical manner as unadjusted data.
To determine the effects of [H+] and temperature on
spore attachment to roots of V. radiata, plants were
inoculated as in the previous experiment except that
temperature and [H+] were altered during inoculation. After
inoculation roots were rinsed for 30-40 sec, stained in
0.05% trypan blue, and cleared in solvent. The second-order
roots were then dissected and mounted in solvent on
microscope slides. The upper one-half surface of roots was
examined at 200x under bright-field illumination using a
Nikon Optiphot compound microscope. Macroconidia were
counted in 20 fields-of-view, in which 50% of the fields-of-
view were second-order roots with root hairs and 50% of the
fields-of-view were second-order roots without root hairs.
The sum of the counts per all 20 fields-of-view, per root
system, were adjusted to represent the number of
macroconidia attached per linear centimeter of root.

110
To determine the effects of temperature and [H+] on
germ tube elongation, plants were inoculated as was
described above for the spore attachment experiment, except
that all plants were inoculated at 25 C and pH 5.0. After
exposure to inoculum plants were rinsed in fresh nutrient
solution maintained at 25 C and pH 5.0, and then individual
plants were transferred to 600-ml beakers containing 500 ml
of fresh nutrient solution adjusted to various combinations
of temperature and [H+]. Plants were placed under six,
20-watt, fluorescent bulbs at a light intensity of 170-180
/nmol • s-1 *m~2 and incubated for 6 hr. Roots were then
stained, dissected, and mounted on microscope slides as
described previously. The lengths of 30 randomly selected
germ tubes per root system were measured using the
integrated video image analysis system, as was described
previously for the in vitro experiments.
A split-plot experimental design, in which temperature
(15, 20, 25, 30, and 35 C ± 1.0 C) was the main plot and
[H+] (pH 3.0, 4.0, 5.0, 6.0, and 7.0 ± 0.1) was the split
plot, was used in both experiments. Tests of each
combination of temperature and [H+] were conducted three
times. Data from both experiments were transformed to logs
and then subjected to orthogonal polynomial contrast
analyses. All possible linear, quadratic, cubic, and
quartic interactions for the effects of temperature and [H+]
on spore attachment were tested.

Ill
An experiment was conducted to determine the effects of
inoculum density and temperature on the severity of disease.
Seven- to eight-day-old plants, grown in seed germination
trays, were transplanted into 800-ml flasks containing
750 ml of fresh nutrient solution. Plants were held in
place by open-celled, polyurethane foam plugs which were
heat treated and autoclaved, as was described previously
(Chapter 2). Flasks were transferred to temperature control
tanks (76 x 38 x 15 cm) in plant growth chambers (EGC, model
M-13 Plant Growth Chambers; Chapter 2). Temperature of the
plant nutrient solution was regulated by controlling the
water temperature in the tanks via closed-loop recirculating
systems connected to Lauda water baths (Chapter 2).
Compressed air was passed through carbon filters and
distributed to each flask to oxygenate the nutrient
solution. Plants were transplanted 24 hr prior to
inoculation to reduce effects of transplant shock.
Inoculum was prepared from 14-day-old MCLA cultures, as
was described previously (Chapter 2). Inoculum densities of
stock suspensions were adjusted to 8.0 X 104 propagules/ml.
Appropriate volumes of inoculum were added to each of seven
flasks, per inoculum density, per growth chamber to provide
0, 100, 500, 1000, 2000, and 3000 propagules/ml. Two growth
chambers were utilized and maintained at similar
temperatures for each experiment. Temperature was
maintained at 20, 25, and 30 C ± 1.0 C; vapor pressure
deficit was held between 6.0 and 7.0 mm Hg for each

112
temperature. Plants were provided a photon flux of 250-275
nmol's ■‘■•m by a mixture of fluorescent and incandescent
lamps. After all treatments were inoculated, three randomly
selected flasks per growth chamber, treated at the density
of 500 propagules/ml, were sampled to confirm that they had
received the correct amount of inoculum. The pH was
adjusted in each flask to 5.5 just prior to exposure of the
plants to inoculum, but after 24 hr the pH was allowed to
fluctuate between 6.0 and 7.0. Plants were maintained for
14 days and then harvested for shoot and root fresh weights.
A completely randomized, split-plot experimental design
was utilized for experiments with temperature as the main
plot and inoculum density as the split plot. Inoculum
treatments were randomized within each growth chamber.
Temperature was replicated twice during each of two
repetitions of the tests. Data were transformed to logs and
subjected to an orthogonal polynomial contrast analysis.
Coefficients for unequally spaced treatments were calculated
using PROC IML within SAS. Effects of inoculum density and
temperature on root, shoot, total plant, and shoot-root
ratios were tested.
A separate experiment was conducted to determine if the
[H+] during inoculation would affect the severity of disease
caused by F. solani f. sp. phaseoli in V. radiata by
altering the numbers of macroconidia attached to roots.
Experimental units consisted of 4-L tanks and closed-loop
water recirculating systems, as were described previously

113
(Chapter 2). A split plot design, in which [H+] (4.0, 5.0,
6.0, and 7.0) was the main plot and inoculum level (0 and
500 propagules/ml) the split plot, was utilized for this
experiment. Combinations of [H+] and inoculum level were
replicated three times during each of three repetitions of
the experiment. Root and air temperatures were maintained
at 24 C ± 1.5 C; ambient humidity was maintained between 7.0
and 9.5 mm Hg, VPD. Four, 7- to 8-day-old seedlings were
transplanted into each of 24 experimental units arranged on
tables within a 2.5-m x 4.5-m tissue culture cleanroom.
High intensity discharge lamps (HID) were suspended over the
plants, as was described previously (Chapter 3). A mixture
of 250-watt, high pressure sodium and 400-watt, metal halide
lamps provided uniform illumination at 275-300 /xmol• s-1 «m-2
at the tops of the plant canopies. Plants were grown for 14
days and then harvested to determine their fresh and dry
weights. Root samples were taken from two plants per
experimental unit and stained in 0.05% trypan blue, as was
described in Chapter 3. Roots were dissected, mounted on
glass slides, and viewed under bright-field microscopy to
determine if conidiophores were produced in all inoculated
treatments.
Nutrient solutions within experimental units were
adjusted to pH 4.0, 5.0, 6.0, and 7.0 ± 0.2 prior to
transplanting seedlings. Plants were transplanted 24 hr
prior to inoculation to reduce effects of transplant-shock.
The [H+] was readjusted for specific treatments just prior

114
to inoculation. After 24 hr, the [H+] within each
inoculated and noninoculated treatment was adjusted to pH
6.0 and thereafter titrated back to pH 6.0 once per day.
Plants were inoculated by dispensing macroconidia from
14-day-old MCLA cultures into the nutrient solution at a
density of 500 propagules/ml, as was described previously
(Chapter 3).
To determine the fluctuations in the numbers of
propagules of F. solani f. sp. phaseoli detected in the
nutrient solutions of inoculated treatments, one, 1.0-ml
sample was withdrawn from each experimental unit receiving
inoculum on 0, 1, 5, 10, and 14 days after inoculation. The
samples were dispensed onto potato dextrose agar
supplemented with 50 mg of chlortetracycline hydrochloride
and 1.0 ml of tergitol NP-10 per liter of medium (PDATC).
Plants were harvested at 14 days after inoculation and
individual plants measured for shoot and root fresh weights
and dry weights. A randomized block design was utilized in
which blocks were structured to determine if a position
effect was present within the tissue culture room. Data
were transformed to logs and subjected to an orthogonal
polynomial contrast analysis. The experiment was conducted
four times.
Results
The optimum temperature and [H+] for germ tube
elongation of F. solani f. sp. phaseoli macroconidia were

115
28 C and pH 7.0, respectively (Figures 4-1 and 4-2). Germ
tube elongation did not occur, and macroconidia failed
to attach to the surface of NSA, at 35 C, below pH 3.0, and
above pH 10.0. Macroconidia germinated at either, or both,
of the terminal and foot cells between 15-30 C, and at all
[H+] tested between pH 3.0 and 10.0. However, when
germination of macroconidia did occur at 32 C, germ tubes
emerged primarily from the lateral side walls of terminal
and foot cells. The effects of temperature and [H+] on the
radial growth of F. solani f. sp. phaseoli were similar to
the effects on germ tube elongation. Mycelial growth did
not occur at 35 C and below pH 3.0, but did occur at all
other temperatures and [H+] tested. The optimum temperature
and pH for mycelial growth were 28 C and 7.0, respectively.
Macroconidia of F. solani f. sp. phaseoli attached
differentially to distinct morphological root orders of
V. radiata. The numbers of macroconidia attached to the
lower hypocotyls were less than those attached to any of the
root orders (Table 4-1) (P < 0.05). When analyzed on the
basis of the numbers of macroconidia attached per linear cm
of root, the numbers of macroconidia were different among
distinct root orders. The highest number of macroconidia
attached per linear cm of root occurred on second-order
roots possessing root hairs. When data were adjusted to
represent spore attachment on the basis of area, and to
compensate for the surface area of root hairs and the
differences in root order diameters, the numbers of

GERMTUBE LENGTH (/¿m)
116
Figure 4-1. Effects of temperature on radial growth and
germ tube length of Fusarium solani f. sp. phaseoli.
Treatment values represent the means of 20 replicates per
treatment for radial growth and three replicates per
treatment for germ tube length; bars indicate standard
deviations of treatment means.
RADIAL GROWTH (mm)

117
Figure 4-2. Effects of hydrogen ion concentration on radial
growth and germ tube length of Fusarium solani f. sp.
phaseoli.
RADIAL GROWTH

118
Table 4-1. Attachment of macroconidia of Fusarium solani f.
sp. phaseoli to four different root orders and to the lower
hypocotyl of Vigna radiata.
Root orders or
hypocotyl
segments
Numbers
of macroconidia
Spores/cmw
O y
Spores/mm£A
Lower hypocotyl^
12 az
19
a
Second-order roots
with root hairs
396 e
252
d
Second-order roots
without root hairs
131 d
158
c
First-order roots
61 c
131
c
Root tips
28 b
83
b
w Data were not adjusted prior to statistical analysis and
represent the numbers of macroconidia counted per linear
centimeter of the root orders or hypocotyl segments.
x Data were adjusted to compensate for differences in area
among root orders and hypocotyl segments attributable to
differences in the diameter of the root and hypocotyl
segments. In addition, data were adjusted to compensate
for the presence of root hairs on the second-order roots
below the crown.
y One-centimeter-long sections of the hypocotyl, directly
above the root crown, were scanned for the presence of
macroconidia.
z Table values represent the means of 15 replicates (three
replicates per treatment in each of five experiments).
Data were transformed to logs and subjected to an Analysis
of Variance followed by Fischer's Least Significant
Difference test; table values are presented as
detransformed numbers. Treatments in columns followed by
the same letter were not different (P > 0.05).

119
macroconidia attached to second-order roots without root
hairs and first-order roots were not different (P > 0.05)
(Table 4-1). However, the numbers of macroconidia
attached to second-order roots with root hairs remained
higher than those for all other root orders
(P < 0.05).
Attachment of macroconidia of F. solani f. sp. phaseoli
to second-order roots of V. radiata was greatest at 20 C and
pH 4.0, although attachment at 25 C was not significantly
different from that at 20 C; spore attachment was reduced
(P < 0.05) as temperature or [H+] were either increased or
decreased (Table 4-2). The numbers of macroconidia attached
per linear cm of root decreased by nearly two orders of
magnitude when the temperature was raised to 35 C or the pH
was elevated to 7.0. The response surface of the
temperature-by-[H+] interaction was best described by a
quadratic-by-quadratic polynomial equation (P < 0.01).
There was no effect of temperature at pH 7.0, nor was there
an effect of the [H+] at 35 C. The effect of temperature
was greatest at pH 4.0 and 5.0 (P < 0.01), and the effect of
[H+] was greatest at 20 and 25 C (P < 0.01). Macroconidia
were misshapen and their internal cell structures disrupted
at pH 3.0, but they were normal at all other [H+].
Macroconidia appeared to attach to the following rhizoplane
sites, in decreasing order of abundance: root hairs,
mucigel, sloughed peripheral rootcap cells (described by

Table 4-2. Effects of temperature and hydrogen ion concentration on the attachment of
macroconidia of Fusarium solani f. sp. phaseoli to second-order roots of Viana radiata.
Temperature
(C)
Numbers
of macroconidia
at
PH
3
.0
4
. 0
5.0
6
.0
7
. 0
15
13
A, az
128
C, b
174 D,b
94
B, b
14
A, a
20
20
A, a
587
D, d
227 C,c
111
B, b
8
A, a
25
63
B, b
355
D, cd
166 C,b
101
C, b
11
A, a
30
45
A, a
320
C, c
188 BC,b
122
B, b
10
A, a
35
7
A, a
7
A, a
3 A, a
5
A, a
3
A, a
z Table values represent the means of nine replicates (three replicates per treatment in
each of three experiments) and correspond to the numbers of macroconidia attached per
linear centimeter of second-order roots. Data were adjusted with a 0.25-power
transformation and then subjected to an orthogonal polynomial contrast analysis; table
values are presented as detransformed numbers. Treatments in rows followed by the same
capital letter, and treatments in columns followed by the same small letter, were not
different based on least-squares mean separation tests (P > 0.05).

Hawes (92)), junction points for the emergence of first-
order roots, root epidermis, and root tips.
121
In preliminary tests, macroconidia on the root surface
began to germinate between 2 and 3 hr after inoculation, and
greater than 90% of macroconidia germinated within 6 hr.
The elongation of germ tubes of macroconidia on the root
surface of V. radiata was greatest at 25 C and pH 5.0, 6.0,
and 7.0 (Table 4-3). Macroconidia failed to germinate at
35 C and at 15 C and pH 3.0. At pH 3.0, as compared to
higher levels, germination was also suppressed at 20-30 C.
Germ tube elongation was generally not affected by [H+]
between pH 5.0 and 7.0 at 15, 20, 25, and 30 C, but was
dramatically affected by temperature within each [H+]
tested. The response surface of the temperature-by-[H+]
interaction was best described by a guartic-by-cubic
polynomial equation (P < 0.01). Germ tubes were distorted
and variable in shape in treatments at pH 3.0 incubated at
20 and 25 C. Under these conditions, germ tubes also tended
to emerge from lateral walls of terminal or foot cells;
these results were consistent with in vitro experiments in
which macroconidia were incubated at pH 3.0.
In the germ tube elongation experiment, most
macroconidia detached from second-order roots when plants
were incubated after inoculation at 35 C or pH 7.0. The
numbers of macroconidia attached to second-order roots were
dramatically higher when plants were incubated at lower
temperatures and hydrogen ion concentrations. The

Table 4-3. Effects of temperature and hydrogen ion concentration on germ tube elongation
of Fusarium solani f. sp. phaseoli after spore attachment to second-order roots of
Viqna radiata.
Temperature
(C)
Germ
tube lengths (/¿m)
at pH
3.0
4.0
5.0
6.0
7.0
15
0.0
A, az
16.5
B, b
20.1 B,b
27.2
C, b
21.0
B, b
20
12.8
A, b
67.0
B, c
00
•
o
0
87.7
C, c
87.0
C, c
25
15.4
A, c
110.0
B, e
140.3 C,e
141.2
C, e
134.7
C,e
30
0.4
A, a
80.8
B, d
101.3 C,d
103.8
C, d
103.6
C, d
35
0.0
A, a
0.0
A, a
0.0 A,a
0.0
A, a
0.0
A, a
z Table values represent the means of nine replicates (three replicates per treatment in
each of three experiments) collected as 30 measurements per replicate. Data were
transformed to square roots and then subjected to an orthogonal polynomial contrast
analysis; table values are presented as detransformed numbers. Treatments in rows
followed by the same capital letter, and treatments in columns followed by the same
small letter, were not different based on least-squares mean separation tests
(P > 0.05).

123
conditions for spore attachment in the germ tube elongation
experiment, 25 C and pH 5.0, were identical for all
treatments. Presumably, similar numbers of macroconidia
attached to second-order roots during plant inoculation,
and, thus, the reduction in the numbers of macroconidia
observed at 35 C and pH 7.0 in the germ tube experiments
represented a loss of macroconidia from the rhizoplane of V.
radiata during the 6 hr post-attachment period.
Effects of inoculum density on plant fresh weight were
observed at 20 and 25 C, but not at 30 C (Figure 4-3). At
20 C, fresh weights of plants inoculated at 100, 500, and
1000 propagules/ml were different from each other and from
all other treatments (P < 0.05). Fresh weights of plants
inoculated at 2000 and 3000 propagules/ml were not different
(P > 0.05). At 25 and 30 C, plant fresh weights at 100
propagules/ml were not different from those in the
noninoculated controls (P > 0.10). At 25 C, fresh weights
of plants inoculated at 500, 1000, and 2000 propagules/ml
were different from each other (P < 0.01). Fresh weights of
plants inoculated at 2000 and 3000 propagules/ml were not
different (P < 0.05). Plant fresh weights are presented
(Figure 4-3) for simplicity; plant dry weights and shoot-
root ratios are presented in Appendix B.
The numbers of plants that wilted by 14 days after
inoculation were greater at 20 C than at 25 C and increased
with increasing inoculum density (Table 4-4). At 30 C, no

PLANT FRESH WEIGHT (g)
124
INOCULUM DENSITY (PROPAGULES/ml)
Figure 4-3. Effects of inoculum density of Fusarium solani
f. sp. phaseoli and temperature on the severity of disease
in Vigna radiata. Bars indicate standard errors of the
means.

125
Table 4-4. Effects of temperature and inoculum density on
the percentages of wilted Viana radiata plants infected by
Fusarium solani f. sp. phaseoli .
Inoculum
density
(propagules/ml)
Percentage
of wilted plants <
at
20 C
25 C
30 C
0
0
A, az
0
A, a
0
A, a
100
47
B, b
0
A, a
0
A, a
500
67
C, c
18
B,b
0
A, a
1000
89
C, d
43
B, c
0
A, a
2000
96
C, d
57
B, c
0
A, a
3000
89
C, d
71
B, c
0
A, a
z Table values represent the means of four replicates (two
replicates in each of two experiments). Data were
transformed to the arcsines of square roots and then
subjected to an orthogonal polynomial contrast analysis;
table values are presented as detransformed numbers.
Least-squares mean separation tests were used to examine
pairwise comparisons between appropriate treatments.
Treatments in rows followed by the same capital letter,
and treatments in columns followed by the same small
letter, were not different (P > 0.05).

126
plants wilted at any of the inoculum densities tested.
However, plant wilt was not considered an acceptable measure
of the severity of disease because wilted plants at 25 C
would often recover from wilt by initiating lateral roots
from the lower stem just above symptomatic tissue.
To determine the effects of [H+] during inoculation on
subsequent disease, the pH was maintained in separate 4-L
plastic tanks at 4.0, 5.0, 6.0, and 7.0 for 24 hr after
inoculation and then adjusted to pH 6.0. Treatments were
then maintained at pH 6.0 for the duration of the
experiment. Fresh weights of inoculated plants were less
than weights of noninoculated controls at pH 4.0, 5.0, and
6.0, but not at pH 7.0 (P < 0.05) (Table 4-5). The effect
of [H+] on the severity of disease was greatest at pH 4.0,
at which fresh weights of inoculated plants were 54-61% less
than those of inoculated plants at pH 5.0 and 6.0. Effects
of inoculation and a [H+]-by-inoculation interaction were
best described by linear polynomial equations (P < 0.002).
The numbers of propagules of F. solani f. sp. phaseoli
detected in nutrient solution were estimated to determine if
the [H+] maintained during inoculation altered the
subsequent production of secondary inoculum. At day zero,
fewer propagules (colony-forming units/ml) were detected
from treatments in which plants were inoculated at pH 4.0
and 5.0 than from treatments in which plants were inoculated
at pH 6.0 and 7.0 (P < 0.05) (Table 4-6). In addition, the
numbers of propagules detected in nutrient solution

127
Table 4-5. Effects of hydrogen ion concentration at the
time of inoculation on disease caused by Fusarium solani f.
sp. phaseoli in Vigna radiata.
PH
Plant
fresh weight (g)
Noninoculated
Inoculated
4.0
10.35 az
3.81* a
5.0
10.54 a
7.09* b
o
•
VO
10.51 a
7.00* b
7.0
9.38 a
8.64 b
z Table values represent the means of nine replicates per
treatment (three replicates per treatment in each of three
experiments). Untransformed data were subjected to an
orthogonal polynomial contrast analysis. Least-squares
mean separation tests were used to examine pairwise
comparisons between appropriate treatments. Significant
differences in comparisons between noninoculated and
inoculated plants at each hydrogen ion concentration are
indicated by an asterisk (*) (P < 0.05). Treatments in
columns followed by the same letter were not different
(P > 0.05) .

Table 4-6. Effects of hydrogen ion concentration at the time of inoculation on the
numbers of propagules of Fusarium solani f. sp. phaseoli detected in nutrient solution
over time in experiments on disease development in Viqna radiata.
PH
No.
of propagules
(cfu/ml)/day
0
1
5
10
14
4.0
452
A, az
1
C, a
2
C, a
5
C, a
23 B, a
5.0
483
A, a
6
B, a
1
C, a
2
C, a
6 B, a
6.0
525
A, b
6
B, a
3
B, a
3
B, a
6 B,a
7.0
521
A, b
20
B, b
17
B, b
13
B, b
14 B,a
z Table values represent the means of nine replicates per treatment (three replicates per
treatment in each of three experiments). Data were transformed to square roots and then
subjected to an orthogonal polynomial contrast analysis when testing the effects of
hydrogen ion concentration within each day and an Analysis of Variance procedure when
testing the effect of day within each hydrogen ion concentration; table values are
presented as detransformed numbers. Treatments in rows followed by the same capital
letter, and treatments in columns followed by the same small letter, were not different
based on least-squares mean separation tests (P > 0.05).

129
increased at day 14 in treatments at pH 4.0 and 5.0, but not
at pH 6.0 and 7.0 (P < 0.05). Sporodochia, large highly
branched conidiophores, and macroconidia were observed in
greatest abundance on roots initially treated at pH 4.0
during inoculation. Conidiophores and macroconidia were
also observed on plants inoculated at pH 5.0 and 6.0, but
their numbers were lower. Sporodochia or highly branched
conidiophores were not observed on roots treated at pH 7.0
during inoculation; unbranched monophialides were observed
emanating from rhizosphere hyphae at pH 7.0, but they were
rare.
Discussion
In soil, attachment of phytopathogenic fungi to root
surfaces may not be a prerequisite for pathogenesis because
propagules may be able to maintain a close association with
roots by their presence in the soil matrix. A fungal
propagule in soil must rely on an adequate inoculum
potential to allow rapid germination, germ tube growth
through soil, and infection of the host when root exudates
are produced (43). However, in a hydroponic nutrient
solution, nonmotile propagules must interact with root
surfaces directly, establishing a close association with the
host. In this manner, attachment of nonmotile propagules to
root surfaces in nutrient solution may be similar to spore
attachment to aerial plant surfaces. Root pathogens in
hydroponic systems that are capable of rapid attachment,

germination, and penetration should have a competitive
advantage in causing disease.
130
In the current study, spore attachment by F. solani f.
sp. phaseoli on roots of V. radiata was found to be affected
by attachment site, temperature, and [H+]. Macroconidia
attached preferentially to second-order roots with root
hairs; differences among root orders and the hypocotyl were
significant even when the data were adjusted to compensate
for variations in the surface areas of separate root orders.
The optimum set of conditions for spore attachment was found
to be 20 C and pH 4.0; as temperature and [H+] were either
lowered or elevated, spore attachment declined. Spore
attachment by F. solani f. sp. phaseoli on roots of
V. radiata appears to be an active process, rather than
simply physical entrapment of macroconidia on root
projections.
Mechanisms that might be involved with attachment of
fungi to plant surfaces have been discussed in recent
reviews (49,53,161,162). Hydrophobic interactions (162);
secreted fungal adhesives (53,161,162); lectins (162);
adsorptive processes, including surface charge phenomena
(49); and protein- or glycoprotein-mediated interactions
(49,53,161) appear to have received the greatest attention.
However, most studies have been on fungal attachment to
foliar and stem surfaces or on the attachment of motile
zoospores to plant roots (49,53,161). There is an extreme

paucity of information on mechanisms of attachment by
nonmotile fungal spores to plant roots.
131
Hydrophobic bonding has been implicated as a mechanism
of spore attachment with Magnaporthe grísea Barr
(Pyricularia orvzae Cav.) on rice (88), Colletotrichum
lindemuthianum (Sacc. & Magn.) Lams. & Scrib. on bean (237),
and Nectria haematococca Berk. & Br. (anamorph: F. solani
f. sp. cucurbitae Snyd. & Hans.) on zucchini squash (97).
However, in the current study, hydrophobic interactions were
not believed to mediate spore attachment by F. solani f. sp.
phaseoli due to the inability of macroconidia to attach to
the hydrophobic surfaces on hypocotyls of V. radiata.
Secretion of adhesive materials has been reported for
Pvthium aphanidermatum (Edson) Fitzp. and P. dissotochum
Drechsler in hydroponic nutrient solution (77) and for
Phvtophthora cinnamomi Rands in water (98,178). In
addition, Marchant and White (136) described the secretion
of a spore mucilage from macroconidia of F. culmorum (Smith)
Sacc. that covered the outer surface of each conidium. The
adhesive materials were apparently secreted directly through
the cell walls of the spores (77,83,98,136). In contrast,
Hamer et al. (88) described the release of a gelatinous
material by the terminal cells of conidia of M. grisea that
appeared to be the result of the disruption of the conidium
walls during hydration of the spores.
The secretion of spore mucilage by macroconidia of
F. solani f. sp. phaseoli was shown not to correlate in time

132
with spore attachment to plant roots (Chapter 5). High
numbers of macroconidia attached to plant roots within 5 min
of inoculation, but the spore mucilage was not observed
before 1 hr (Chapters 5 and 6). However, results from the
current study, and from Chapters 5 and 6, do not eliminate
the possibility that the rapid secretion of a thin layer of
adhesive material was involved in spore attachment. Rapid
secretion of adhesive materials from fungal spores has been
reported (77,82,88,123).
The functional components of adhesive materials may
include lectins (162). Lectins have been described in roots
and hypocotyls of V. radiata (90,114), reported on root
hairs of other legumes (54,74), and implicated as a
functional component in fungal spore attachment to plant
surfaces (1,73,91,98,130). In addition, conidia of
F. solani f. sp. phaseoli can be agglutinated by the lectin
concanavalin A (Con A) (47,118), suggesting that exposed
sugar moieties on the surface of macroconidia might be
involved with attachment to plant lectins. However, the
sugar hapten of Con A, as well as six other sugar haptens of
lectins, failed to prevent attachment of F. solani f. sp.
phaseoli macroconidia to roots of V. radiata (Chapter 6).
These studies support the conclusion that a chemical binding
phenomenon, other than lectins, may have been involved in
spore attachment of F. solani f. sp. phaseoli to V. radiata.
However, the possibility that a previously undescribed

133
lectin was involved in spore attachment should also be
considered.
Adsorption to plant surfaces is another mechanism that
might be involved in spore attachment by F. solani f. sp.
phaseoli. Daniels (49) listed 16 attractive and four
repulsive forces that could occur between microbial cells
and adsorbent surfaces; surface-charge characteristics were
components in over half of these forces. In addition, the
sorption environment can be greatly affected by [H+] and the
presence of inorganic ions or complex organic compounds that
alter surface charges (49). Adsorption can often be
reversed by changing the [H+] , which suggests the
possibility that complete charge reversals occur (49). In
the current study, extremes of pH near 3.0 and 7.0 greatly
reduced the numbers of macroconidia attached to root
surfaces. In addition, macroconidia that bound to root
surfaces at 25 C and pH 5.0 during plant inoculations in the
germ tube elongation experiment detached from roots when
plants were transferred to the extremes of temperature and
[H+] tested. Adsorption may also be reduced when ions
involved in cation bridges are rendered unavailable at
neutral or alkaline [H+] (41). Although results from the
current study are consistent with many attributes of
adsorptive processes, adsorption may not be the sole
mechanism involved in spore attachment.
The suppression of spore attachment by F. solani f. sp.
phaseoli at high temperature (35 C) and low acidity (pH 7.0)

134
also supports the hypothesis that the binding properties of
the host-fungus interaction may involve proteins, or protein
moieties in larger macromolecules. Extremes of temperature
and [H+] can change the stoichiometry of proteins, alter the
surface charge of macromolecules, or induce denaturation of
proteinaceous compounds (126). In addition, respiration and
protein synthesis have been shown to be required for the
attachment of macroconidia of N. haematococca (112).
Proteins have been implicated as the adhesive material in
hyphopodia of the marine fungus, Buergenerula spartinae
Kohl. & Gess. (167), and in the bean rust fungus, Uromvces
appendiculatus (Pers.) Unger (63). Glycoproteins have been
reported to be a component in adhesive secretions from other
fungal plant pathogens (83,200).
A neutral or slightly alkaline [H+] may also interfere
with spore attachment by altering the availability of
micronutrients in the infection court. Effective
suppression of Fusarium oxvsporum (Schlecht.) f. sp.
lycopersici (Sacc.) Snyd. & Hans, on Lvcopersicon esculentum
(L.) Mill, by the addition of lime to soils has been
demonstrated (109,110,111); the micronutrient deficiencies
created by alkaline conditions were suggested as the
mechanism of disease suppression (109). A neutral or
slightly alkaline [H+] has also been shown to suppress root
disease caused by Plasmodiophora brassicae Wor. (143) and
Phvtophthora cinnamomi Rands (19) in hydroponic systems.
These results are in agreement with Diehl and Steadman's

135
(55) study, in which the incidence of root rot caused by
F. solani f. sp. phaseoli on Phaseolus vulgaris L. was
reduced by increasing the soil pH from 4.9 to 7.8. At [H+]
greater than pH 6.5, Fe3 + and Ca2+ ion species begin to
precipitate from nutrient solution (41). Calcium has been
implicated as a required cofactor in the adsorption of
Fusarium moniliforme Sheldon and Phialophora radicicola Cain
to root mucilage of Zea mays L. (80), and it has been shown
to enhance the agglutination of macroconidia of F. solani f.
sp. phaseoli and F. oxvsporum in the presence of two lectins
(47). Elad et al. (60) suggested that calcium and other
divalent cations may be required for lectin activity. Iron
has been implicated as an essential ion for spore
germination by Fusarium spp. in the rhizosphere
(58,59,195,196), although the effects of Fe on fungal
attachment to plant roots were not studied.
Competition for Fe has also been implicated in the
control of Fusarium wilt of carnations grown in a rockwool
hydroponic system (224). Addition of a siderophore
producing strain of Pseudomonas sp. and the Fe-chelate,
Fe-ethylenediaminedi-O-hydoxyphenyl-acetic acid (Fe-EDDHA),
reduced disease by limiting Fe to the pathogen.
Micronutrients may be involved directly in the binding
phenomenon, as implicated by their role as cation bridges in
adsorption (49), or they may be required by physiologically
active propagules to germinate or infect host tissues. In
either case, their availability in soils, and in hydroponic

136
nutrient solutions, can be reduced by neutral or slightly
alkaline [H+].
The effect of nitrogen source on spore attachment to
roots may also alter the infection court environment by
altering the [H+] in the rhizosphere. Elmer (62)
demonstrated that nitrate-nitrogen, compared to ammonium-
nitrogen, reduced disease in asparagus caused by Fusarium
spp. The effect was enhanced by concomitant treatment with
NaCl or KC1 (62). One mechanism for the increase in disease
with ammonium treatments might have been the stimulation of
spore germination, as was described for chlamydospores of
F. solani f. sp. phaseoli (44). An alternative mechanism
might involve altering the [H+] in the infection court by
changing the nitrogen source, which might then alter spore
attachment to root surfaces. Nitrate-fertilizer increases
the pH, and ammonium-fertilizer decreases the pH in the
rhizosphere as plants attempt to maintain an electro¬
chemical balance within the root symplast during the
assimilation of NO3- or NH4 + (138). The implications of the
study by Elmer (62) are even more interesting when the
abilities of salts to increase desorption of microorganisms
from surfaces (49) are considered. The form of nitrogen
used in hydroponic nutrient solution is critical for
determining the magnitude and the method for [H+] control.
Calcium nitrate and potassium nitrate were recommended by
Hoagland and Arnon (99) in their widely accepted formula for
a hydroponic nutrient solution. If the [H+] is not actively

137
controlled when using nitrate-nitrogen, the pH will rise
sharply (103), often exceeding pH 7.5 within a few days
(Schuerger, unpublished). Further research is required to
discern if different nitrogen sources might alter fungal
attachment to plant roots in hydroponic systems by affecting
the [H+] on the rhizoplane.
The mechanism that mediates spore attachment of
F. solani f. sp. phaseoli to roots of V. radiata is not
known. Results from the current study support both the
adsorptive and protein-mediated mechanisms, but the possible
effects of neutral or slightly alkaline [H+] on nutrient
availability must also be considered. Additional research
is required to fully characterize the mechanism of spore
attachment by F. solani f. sp. phaseoli to plant roots.
Future research should also consider the possibility that
separate mechanisms might be involved in spore attachment.
Although it seems reasonable to expect a single mechanism to
be operating, Nicholson and Epstein (162) concluded that
there is no evidence in the literature to dispel the
possibility that several binding mechanisms are
concomitantly involved in spore attachment.
The significance of root diseases in hydroponic systems
has been questioned by several researchers (51,72,100,
177,212). Certain hydroponic systems may have inherent
qualities that suppress disease, as some have suggested
(100,177,184), but it seems that this position should be

138
retained with caution until more detailed studies on the
effects of [H+] and temperature on disease can be conducted.
Based on the results of the spore attachment
experiment, and on pathogenicity tests conducted at
different combinations of temperature and [H+] (Chapter 3),
it was hypothesized that the effects of [H+] on disease
would differ depending on when the [H+] treatments were
applied. When the [H+] was adjusted after plant
inoculation, pH 4.0 was found to reduce the severity of
disease caused by F. solani f. sp. phaseoli in V. radiata
(Chapter 3). In addition, when pH was maintained at 4.0
after inoculation, it was found to suppress the production
of secondary inoculum at 20 C, a temperature normally
conducive to the production of secondary macroconidia. In
the current study, when [H+] was adjusted during plant
inoculation, pH 4.0 was found to enhance the severity of
disease and increase the production of secondary inoculum at
25 C, a temperature that had previously been shown to have a
minor or nonexistent effect on the production of secondary
macroconidia (Chapter 3). Additionally, the high level of
disease observed when pH was maintained at 4.0 during
inoculation correlates with the high number of spores
attached to roots of V. radiata when the pH was maintained
at 4.0 during the spore attachment experiment. These
results support the conclusion that the timing of a [H+]
treatment can affect separate aspects of pathogenesis.
Post-attachment and post-infection processes of pathogenesis

139
were altered when plants were maintained at different [H+]
after inoculation, but spore attachment and host infection
processes of pathogenisis were altered when plants were
inoculated at different [H+].
Results of the inoculum density study confirm that
disease severity increases with increasing levels of
inoculum in a hydroponic system. Effects of inoculum
density were observed at 20 and 25 C, but not 30 C, which is
consistent with the effects of temperature on disease caused
by F. solani f. sp. phaseoli in V. radiata observed in
Chapter 3. A distinct flattening of the disease and
inoculum density curves was observed at 20 and 25 C. This
response is interpreted to indicate a saturation of
attachment or infection sites at high inoculum densities, as
was described by Vanderplank (222) .
The percentage of wilted plants at 14 days decreased as
temperature increased and increased as inoculum density
increased at 20 and 25 C. Plant wilt was positively
correlated to disease in the current study; plant wilt was
highest under environmental conditions that promoted the
development of disease. However, based on previous studies
(Chapter 3) plant wilt was not considered an acceptable
method for evaluating disease severity.
Results of the current study also bring into question
the procedures utilized to inoculate plants with Fusarium
spp. by submerging their roots in spore suspensions.
Conditions of nutrient composition, temperature, and [H+] in

140
the spore suspensions were not presented in any of the
papers reviewed (8,21,125,142,171). Although the procedure
is utilized effectively, it would be interesting to discern
the effects of these parameters on spore attachment. High
densities of spores, often between 105 and 107
propagules/ml, were utilized for root-dip inoculations in
these studies. In contrast, a density of 500 propagules/ml
was adequate to produce severe disease in the F. solani f.
sp. phaseoli and V. radiata pathosystem at 20 and 25 C. The
differences in the functional inoculum densities among these
studies may be greatly influenced by the effects of
temperature or [H+] on spore attachment. It also may be
possible that microconidia of Fusarium spp. are not as
adhesive as either macroconidia or chlamydospores;
microconidia were the exclusive infective propagule in most
of the studies reviewed (8,21,125,142). Additional research
is required to study the effects of temperature and [H+] on
the attachment of microconidia, macroconidia, and
chlamydospores of Fusarium spp. to root surfaces.

CHAPTER 5
THE INFLUENCE OF SECRETED SPORE MUCILAGE OF MACROCONIDIA OF
FUSARIUM SOLANI F. SP. PHASEOLI ON SPORE ATTACHMENT TO ROOTS
OF VIGNA RADIATA IN HYDROPONIC NUTRIENT SOLUTION
Introduction
The soilborne, pathogenic fungus, Fusariuin solani
(Mart.) Appel. & Wr. f. sp. phaseoli (Burk.) Snyd. & Hans.,
is capable of infecting both hypocotyl and root tissues of
bean plants grown in soil (32,34,38,120); plant ingress can
be either through stomata or via direct penetration of host
tissues (39,173). In contrast, F. solani f. sp. phaseoli
caused few hypocotyl lesions when seedlings of Viana radiata
(L.) Wilczek were inoculated in a hydroponic nutrient
solution (Chapter 3). In soil, infective propagules of
nonmotile phytopathogenic fungi may be maintained in a
position favorable for infection of plant tissues by the
soil matrix. In hydroponic nutrient solution, infective
propagules of nonmotile fungal pathogens must be able to
attach to root tissues directly before pathogenesis can be
initiated. If spore attachment is impaired, plant infection
and disease will likely be reduced. Disease caused by
F. solani f. sp. phaseoli in V. radiata was reduced when
plants were inoculated at pH 7.0, compared to those
inoculated at pH 4.0-6.0; the lower level of disease was
141

142
attributed to a reduction in the numbers of infective
propagules attached to root surfaces at pH 7.0 (Chapter 4).
Various mechanisms have been proposed that might
mediate fungal attachment to plant surfaces (49,53,161,162).
The secretion of fungal adhesives has been reported to be
involved in the attachment of motile zoospores of
Phytophthora spp. (83,200) and Pvthium spp. (77,82) to root
surfaces, and in the attachment of nonmotile conidia of
Maqnaoorthe qrisea Barr (88) and Cladosporium cucumerinum
Ell. & Arth. (172) to foliar and hypocotyl surfaces. The
timing of the release of adhesive materials appeared to be a
prerequisite for spore attachment. In addition, different
mechanisms may be utilized by fungal propagules for the
release of the adhesive materials. For example, the
secretion of spore mucilage by zoospores of Pythium
aphanidermatum (Edson) Fitzp. occurred during zoospore
encystment, and required no more than 2-3 min (82) . In
contrast, spore mucilage was released from the spore tips of
M. qrisea by the rupture of the conidium walls during
hydration (88); release of the spore mucilage was rapid and
independent of spore contact with a surface.
The secretion of spore mucilage by several Fusarium
spp. has been reported (118,135,136), but no information was
found in the literature on whether specific timing of
secretion of spore mucilage by a Fusarium sp. was essential
for spore attachment to plant surfaces. The objectives of
this study were 1) to determine if the timing and location

143
of the secretion of spore mucilage were significant factors
in the attachment of macroconidia of F. solani f. sp.
phaseoli to roots of V. radiata, and 2) to determine if the
combinations of temperature and hydrogen ion concentration
that suppressed spore attachment in a previous study
(Chapter 4) would also suppress the secretion of spore
mucilage.
Materials and Methods
Effects of culture age on the cytology of macroconidia
of Fusarium solani f. sp. phaseoli were evaluated.
Macroconidia of isolate F-28A (obtained from A. J. Anderson,
Utah State University, Logan, Utah) were collected in
sterile deionized water (SDIW) from 7- to 102-day-old
modified carnation leaf agar (MCLA) cultures (Chapter 2).
Spores were stained for 5 min in 100 ¡jl 1 of a 0.05% trypan
blue stain (Chapter 2), transferred to the surface of
nutrient solution agar (NSA) (Chapter 2), overlaid with
coverslips, and then photographed with Polaroid® 4x5 Instant
Film, Type N55 (Polaroid Corp., Cambridge, MA, 02139) using
a Nikon Optiphot compound microscope.
In preliminary experiments using scanning electron
microscopy (SEM) to study the attachment of macroconidia to
roots of V. radiata. 14-day-old macroconidia appeared to be
poorly fixed, which resulted in the distortion and collapse
of the macroconidium walls. To determine the effects of
culture age on spore fixation, macroconidia were grown on

144
MCLA for 7, 10, 12, 14, and 21 days. Spores were prepared
for SEM using two different methods for fixation. Fixation
and buffer rinses were conducted at 4 C. In the first
procedure, carnation leaf pieces possessing abundant
conidiophores and macroconidia were fixed in 3.0%
glutaraldehyde (Ted Pella, Inc, Tustin, CA, 92680) in
Sorensen's phosphate buffer (79) at pH 6.8 for 4 hr.
Samples were rinsed three times at 20-min intervals in fresh
buffer and then post-fixed in a similarly buffered 1.0%
osmium tetroxide solution (Ted Pella, Inc, Tustin, CA,
92680) for 12-18 hr. Samples were then rinsed three times
in buffer at 20-min intervals. Following the final buffer
rinse, samples were washed three times at 20-min intervals
in double-deionized water at 4 C; samples were then allowed
to warm to room temperature (approximately 23-25 C). In the
second procedure, macroconidia of similar ages were fixed
for 18-24 hr in a 1.0% unbuffered osmium tetroxide solution
containing 0.003% Photo-flo (Eastman Kodak Company,
Rochester, NY, 14650), as was described by Brown and
Brotzman (28). Samples from both fixation procedures were
dehydrated in 10% incremental ethanol series. Carnation
leaf pieces were critical-point dried using CO2 and then
coated with gold in a Ladd 30800 sputter coater (Ladd
Research Industries, Inc., Burlington, VT, 05402). Samples
were photographed with Polaroid 4x5 Instant Film using a
Hitachi S-530 scanning electron microscope (Hitachi
Instruments, Inc., Danbury, CT, 06810).

145
Agglutination of macroconidia of F. solani f. sp.
phaseoli was determined in four solutions prepared from
components of the plant-hydroponic system. A root leachate
solution was prepared by incubating 40-50, 7- to 8-day-old
seedlings of V. radiata in 600 ml of nutrient solution. The
production of seedlings and the composition of the nutrient
solution have been described previously (Chapter 2). Plants
were maintained for 18 hr at room temperature. The nutrient
solution was aerated continuously using filter-sterilized
compressed air, as was described previously (Chapter 2). A
root-homogenate solution was prepared by triturating 10 g
(fresh weight) of 7- to 8-day-old V. radiata roots in 30 ml
of nutrient solution; roots were severed 5 mm below the
crown. The root-homogenate extract was diluted in 1 L of
nutrient solution and allowed to stand at room temperature
for 30-60 min. Double deionized water and fresh nutrient
solution were used as control treatments. The root leachate
and root homogenate preparations were filtered through four
layers of cheesecloth, and then all solutions were filtered
through 0.45-jum, Gelman Mini-Capsule, cartridge filters
(Gelman Sciences, Inc., Ann Arbor, MI, 48106). Prior to
specific tests root-leachate, root-homogenate, and control
solutions were titrated to pH 5.5. The hydrogen ion
concentration ([H+]) was adjusted in this and all subsequent
experiments using 0.01 N HNO3 and 0.02 N KOH. Macroconidia
were washed from 14-day-old MCLA cultures using 40 ml of
SDIW. Macroconidia were washed three times by first

146
collecting them on a 5.0-/im, cellulose nitrate filter (MSI
Micron Separations, Inc., Westboro, MA, 01581) and then
resuspending them in 40 ml of SDIW. Spore agglutination was
determined by dispersing washed macroconidia of F. solani f.
sp. phaseoli into 50 ml of each test solution. Spore
densities were adjusted to 5-10 X 104 macroconidia/ml. A
hemacytometer was used to estimate the numbers of propagules
in this and all subsequent experiments. After spore
suspensions were gently agitated for 5 hr using magnetic
stirrers, 100-/il samples were withdrawn from each treatment
at 20, 30, 60, 180, and 300 min, mounted on acid-washed
glass slides, and then observed under bright field light
microscopy.
Spore agglutination was considered positive when at
least four aggregates of macroconidia were observed in a
100-/L11 sample; each aggregate was composed of at least four
individual macroconidia, among which spore-to-spore contact
occurred between terminal or foot cells. Acid-washed, glass
slides were essential for observing spore agglutination
because macroconidia adhered to refuse on unwashed slides.
In addition, very low numbers of spore aggregates consisting
of two or three macroconidia were observed in all treat¬
ments, and they were considered a background phenomenon.
To determine the effects of culture age on spore
agglutination, macroconidia were grown on MCLA for
7-102 days, harvested in SDIW, and then dispersed in 50 ml
of root homogenate to achieve a final spore density of

147
between 5 and 10 x 104 macroconidia/ml. Macroconidia were
gently agitated for 1-1.5 hr using magnetic stirrers and
lOO-fil samples were mounted on glass slides and viewed under
bright field microscopy.
Experiments using SEM to study the process of
macroconidium attachment to roots of V. radiata were
conducted using the buffered glutaraldehyde and osmium
tetroxide fixation procedure described above, except that
glutaraldehyde fixation was extended to 12-18 hr. Seven- to
eight-day-old seedlings were transferred to 500 ml of
nutrient solution maintained at 25 C and pH 5.5. Plants
were supported in the nutrient solution using closed-cell,
polyurethane foam plugs which were heat treated and
autoclaved, as was described previously (Chapter 2).
Inoculum was prepared in SDIW using 10- to 12-day-old MCLA
cultures. Inoculum was adjusted to a density of 3-5 x 104
propagules/ml. Roots were inoculated for 5 or 10 min,
briefly rinsed in fresh nutrient solution, and second-order
roots dissected for SEM preparation. In addition, a subset
of dissected roots was stained in 0.05% trypan blue for
5 min, cleared in solvent, mounted on glass slides, and then
viewed under bright field microscopy.
Macroconidia of F. solani f. sp. phaseoli were
incubated in root homogenate at various combinations of
temperature and [H+] to determine the effects of these
variables on spore agglutination. Macroconidia from 10- to
12-day-old MCLA cultures were incubated at a density of

148
5-10 x 104 macroconidia/ml for 1-1.5 hr in 300 ml of
unbuffered root homogenate and then collected on Whatman
No. 42 filter paper. Without permitting the desiccation of
macroconidia, discs of filter paper were transferred to
fresh nutrient solution and then cut into 4.0-mm2 pieces for
fixation. Macroconidia were fixed in unbuffered osmium
tetroxide containing Photo-flo and prepared for SEM, as was
described above. In one experiment, temperature was
maintained at 25 C and separate treatments were adjusted to
pH 3.0, 4.0, 5.0, 6.0, and 7.0. In a second experiment, the
pH was maintained at 5.0 and separate treatments were
adjusted to 15, 25, and 35 C. Root temperatures were
controlled by water baths, as was described previously
(Chapter 4).
All experiments were conducted at least three times.
The experiment on the effects of culture age on spore
agglutination was conducted five times, and the experiments
on the effects of temperature and [H+] on spore
agglutination were conducted four times.
Results
The sizes and numbers of vacuoles increased as the age
of macroconidia of F. solani f. sp. phaseoli increased
(Figure 5-1). Vacuoles were not frequently observed in
7-day-old macroconidia, but they were abundant in
macroconidia older than 47 days. Chlamydospores generally
developed from terminal and foot cells in macroconidia older

Figure 5-1. Effects of culture age on the cytology of
macroconidia of Fusarium solani f. sp. phaseoli. The size
and number of vacuoles (arrows) increased in both apical and
intercalary cells of macroconidia as their age increased.
Few vacuoles were observed in macroconidia from 10-day-old
cultures, but were abundant in 47-day-old cultures.
Chlamydospores (ch) were observed in 87- and 102-day-old
cultures.

150

151
than 60 days. Macroconidia did not appear to change their
overall shape or dimensions as they aged.
Macroconidia older than 12 days exhibited collapsed and
distorted cell walls when prepared for SEM (Figure 5-2).
Macroconidia between 7 and 10 days old were smooth and
uniform in shape. Septation in younger spores was not
observed as easily as it was in spores older than 12 days
(Figure 5-2). Both fixation procedures yielded similar
results.
Spore agglutination was not observed in deionized water
and nutrient solution (Figure 5-3A), but it was observed
periodically in different batches of root leachate. A
strong and consistent agglutination response was observed
when macroconidia of F. solani f. sp. phaseoli were
incubated for 20-30 min in root homogenate (Figure 5-3B
and C). In addition, refuse attached to spore tips when
macroconidia were incubated in root homogenate
(Figure 5-3B). The procedure of washing spore suspensions
using the cellulose nitrate filters dramatically reduced,
but did not eliminate, refuse in subsequent tests.
The age of macroconidia also influenced the intensity
of agglutination when spores were incubated in root
homogenate. Spore agglutination was weakest with
macroconidia from 7- to 12-day-old and 40- and 60-day-old
cultures. The strongest agglutination response in root
homogenate was observed with macroconidia from 14- to
40-day-old MCLA cultures, in which aggregates of several

Figure 5-2. Effects of age on fixation and preparation of
macroconidia of Fusarium solani f. sp. phaseoli for scanning
electron microscopy. Macroconidia from 7-day-old cultures
were smooth and exhibited few signs of distorted or
collapsed surfaces. Macroconidia from 14- and 21-day-old
cultures exhibited surface distortions and prominent septa.

153

Figure 5-3. Agglutination of macroconidia of Fusarium
solani f. sp. phaseoli incubated in nutrient solution and
root homogenate. A, Macroconidia failed to agglutinate when
incubated in nutrient solution, regardless of the
temperature and hydrogen ion concentration. B, Macroconidia
agglutinated in all tests utilizing root homogenate; refuse
(rf) from the modified carnation leaf agar cultures attached
to the terminal and foot cells of macroconidia. C,
Macroconidia formed large aggregates in root homogenate
where spore-to-spore attachment was almost exclusively by
tip-to-tip contact.

155

156
hundred macroconidia were observed (Figure 5-3C).
Macroconidia that were from 87- and 102-day-old cultures
failed to agglutinate in root homogenate.
Macroconidia attached to the following rhizoplane
sites, in decreasing order of abundance: root hairs,
mucigel, sloughed peripheral rootcap cells (described by
Hawes and Pueppke (92)), junction points for the emergence
of first-order roots, root epidermis, and root tips. Spores
attached immediately to root surfaces at their terminal and
foot cells (Figure 5-4), and they were not generally washed
off of roots when roots were rinsed in nutrient solution
after inoculation. Macroconidia appeared to attach to both
intact (Figure 5-4A) and collapsed (Figure 5-4B) root hairs
of V. radiata. Refuse was usually observed on the terminal
and foot cells of macroconidia attached to root surfaces
(Figure 5-4). In addition, macroconidia readily attached to
large islands of mucigel present on root surfaces
(Figure 5-4D).
In a series of experiments, macroconidia of F. solani
f. sp. phaseoli were incubated in root homogenate at various
combinations of temperature and [H+]. Agglutination of
macroconidia was observed at 25 C and pH 4.0, 5.0, and 6.0,
but not at 25 C and pH 3.0 or 7.0 (Figure 5-5). When the pH
was maintained at 5.0 and the temperature varied,
agglutination of macroconidia was observed at 15 and 25 C,
but not at 35 C; macroconidium aggregates were larger and
more numerous at 25 C than at 15 C.

Figure 5-4. Attachment of macroconidia of Fusarium solani
f. sp. phaseoli to root surfaces of Viqna radiata.
Macroconidia readily attached to intact (A) and
collapsed (B) root hairs (rh). A,B,C, Refuse (rf) of
unknown origin was observed on spore tips of macroconidia
within 5 min after plant inoculation; macroconidia attached
to root surfaces at their spore tips. D, Macroconidia
attached to mucigel (m) on the root epidermis (ep).


Figure 5-5. Agglutination of macroconidia of Fusarium
solani f. sp. phaseoli in root homogenate maintained at 25 C
and at different hydrogen ion concentrations. Spore-tip to
spore-tip agglutination (a) was not observed at pH 3.0 and
7.0, but was observed at pH 4.0, 5.0, and 6.0. Fewer
macroconidia adhered to the filter membranes (fm) at pH 3.0
than at all other hydrogen ion concentrations tested.
At pH 7.0, macroconidia bound to nutrient solution
precipitates (np).


161
Refuse attached to macroconidium tips in temperature
and [H+] combinations where spore agglutination was observed
(Figure 5-6). In contrast, refuse was not observed on
macroconidium tips in treatments where spore agglutination
failed to occur. The accretion of refuse on to the apical
cells of macroconidia was only observed in root homogenate
and when spores were attached to roots of V. radiata.
Macroconidia incubated in nutrient solution, SDIW, and root
leachate were free of refuse, regardless of the temperature
or [H+]. Refuse was believed to be mycelium fragments,
fungal cytosol, and shards of carnation leaves concomitantly
harvested with macroconidia from MCLA cultures (Figure
5-6A). Refuse appeared to act as centers for spore
attachment (Figures 5-3 and 5-6). In addition, small
fragments of refuse were observed attached to cell walls of
terminal, intercalary, and foot cells of macroconidia
(Figure 5-6B and C). Terminal and foot cells of F. solani
f. sp. phaseoli macroconidia appeared to be equally
receptive to the attachment of refuse (Figure 5-6C). Refuse
was not observed on spore tips when macroconidia were
incubated at pH 3.0 and 7.0 at 25 C, nor was it observed
when they were incubated at pH 5.0 and 35 C. At 25 C and pH
7.0, salts from the nutrient solution precipitated and
subsequently adhered to all macroconidium surfaces (Figure
5-5E); spore-tip to spore-tip agglutination of macroconidia
was not observed at pH 7.0.

Figure 5-6. Attachment of refuse from cultures of modified
carnation leaf agar (MCLA) to macroconidia of Fusarium
solani f. sp. phaseoli when macroconidia were incubated in
root homogenate. A, Refuse (rf) from MCLA cultures was
collected concomitantly with macroconidia (co = conidio-
phore). B,C, Refuse appeared to act as centers for spore
agglutination; terminal (tc) and foot (fc) cells of
macroconidia appeared equally receptive to the attachment of
refuse.

163

164
An amorphous material, termed spore mucilage, was
secreted by terminal and foot cells of germinating macro-
conidia (Figure 5-7) incubated in root homogenate at 25 C
and pH 4.0-6.0, but not when macroconidia were incubated at
pH 3.0 and 7.0. In a second experiment in which pH was
maintained at 5.0 and temperature adjusted to different
levels, spore mucilage was observed on macroconidia at 15
and 25 C, but not at 35 C. When macroconidia were incubated
in root homogenate, germ tubes emerged primarily from the
apices of terminal and foot cells, and periodically from the
lateral walls of apical or intercalary cells. The spore
mucilage was observed at the sites of germ tube emergence,
but it was not observed along the lengths of the elongating
germ tubes (Figure 5-7A). The spore mucilage had the
physical attributes of a soft viscous material; it appeared
to flow away from spore tips upon contact with other
surfaces (Figures 5-7B and C). Spore mucilage was rarely
observed on macroconidia incubated for 1 hr in root
homogenate, but it was observed on macroconidia incubated
for 1.5 hr in root homogenate. Mucilage was never observed
on the spore tips of macroconidia that had attached to root
surfaces. The accretion of refuse to spore tips did not
appear to require spore mucilage (Figure 5-6B and C);
however, it appeared that refuse could attach to spore
mucilage in treatments incubated in root homogenate (Figure
5-7D).

Figure 5-7. Production of spore mucilage at the tips of
macroconidia of Fusarium solani f. sp. phaseoli during
germination of conidia in root homogenate. A, Spore
mucilage (sm) was observed at the sites of germ tube (gt)
emergence from apical cells of macroconidia (ma); the spore
mucilage was not contiguous with elongating germ tubes.
B,C, The spore mucilage exhibited qualities of a soft
viscous material, flowing away from spore tips upon contact
with other surfaces. D, Spore mucilage also bound to refuse
(rf) present in the spore suspensions. Arrows delimit the
extent of the secreted spore mucilage.

991

167
Discussion
Extracellular adhesive materials have been described on
fungal germlings (65,93,161,162), nongerminated conidia
(88,172) and zoospores (82,83,200). The release of the
adhesive materials appeared to be a prerequisite for spore
attachment with several of these fungal pathogens
(82,88,172,200). In addition, spore mucilages have been
reported on several Fusarium spp. (118,135,136), but the
timing of the secretions to spore attachment were not
studied.
The functional components of adhesive materials may
include lectins, proteins, carbohydrates, or non-lectin
glycoproteins (161,162); hydrophobic interactions (88,97)
and adsorptive processes (49,53) also may be involved in
fungal attachment to surfaces. Various inhibitors have been
tested for their effects on fungal attachment to surfaces.
For example, the addition of concanavalin A (Con A)
interfered with the attachment of conidia of M. grisea to
hydrophobic surfaces (88). Inhibitors of respiration and
protein synthesis reduced conidium adhesion to hypocotyls
and polystyrene by Colletotrichum lindemuthianum (Sacc. &
Magn.) Lams. & Schrib. (237) and Nectria haematococca (Berk.
& Br.) (112). However, only one paper was found in the
literature on the effects of [H+] on adhesion of fungal
propagules to plant roots; conidia of F. moniliforme Sheldon
and Phialoohora graminicola Cain responded similarly to [H+]
with optimum adsorptions to root mucilage at pH 6.5 (80).

168
The pH optimum for attachment of F. solani f. sp. phaseoli
macroconidia to roots of V. radiata was 4.0 (Chapter 4).
Based on these studies, it was hypothesized that if a
secreted spore mucilage is involved in the attachment of
macroconidia of F. solani f. sp. phaseoli to roots of
V. radiata. then its secretion should coincide with spore
attachment under different conditions of temperature and
[H+]. A series of tests were conducted to determine the
effects of temperature and [H+] on spore attachment to
roots, on the agglutination of macroconidia in a crude root-
extract, and in the location and timing of the secretion of
spore mucilage. Macroconidia of F. solani f. sp. phaseoli
immediately attached to root surfaces after plants were
inoculated in nutrient solution. Macroconidia attached to
most surfaces at their spore tips, confirming earlier
studies (Chapters 3 and 4). A secreted spore mucilage was
observed on macroconidia at the time of germination
(approximately 1-1.5 hr) in root homogenate, but it was not
observed during the initial stages of spore attachment to
roots of V. radiata. Spore mucilage was always observed at
the tips of macroconidia and usually preceded the emergence
of germ tubes. Although spore tips were involved in
macroconidium attachment to roots, and as sites for
agglutination in root homogenate, the secreted spore
mucilage may not be the adhesive factor in spore attachment
to roots because it was not observed at the time of spore
attachment. These results do not eliminate the possibility

169
that a rapidly secreted, thin layer of adhesive material was
involved in spore attachment to roots. Grove and Bracker
(82) demonstrated that the secretion of an adhesive cyst
coat by Pvthium aphanidermatum (Edson) Fitzp. occurred in
less than 1 min; the rapid secretion of adhesive materials
has been reported for other fungi (77,123,200).
The secretion of spore mucilage, accretion of refuse to
spore tips, agglutination of macroconidia in root
homogenate, and spore attachment to root surfaces were
similarly affected by changes in temperature and [H+].
These processes were all suppressed at pH 3.0 and 7.0, and
at 35 C, confirming earlier quantitative studies on the
effects of temperature and [H+] on spore attachment by
macroconidia of F. solani f. sp. phaseoli (Chapter 4). It
seems likely that these processes are related because they
respond to temperature and [H+] in similar ways. No reports
were found in the literature on the effects of temperature
or [H+] on spore agglutination in plant extracts or in the
presence of phytoagglutinins. However, Kauss and coworkers
(114,115) described a phytoagglutinin from V. radiata that
was sensitive to changes in [H+]; peak activity of the
phytoagglutinin was between pH 7.0 and 8.0 (114). Based on
the results from the current study, it is proposed that the
secretion of spore mucilage is a later step in the process
of germination that begins by the contact of macroconidium
tips with root surfaces. In addition, it is proposed that
the first step in this process is the adsorption or binding

170
of an inducer to the spore tips. The inducer might be a
component of the root mucilage, a constituent of epidermal
cell walls, or a diffusible extracellular protein or
glcoprotein of plant origin. Agglutinins have been
described on root mucilage (80), in hypocotyl cell walls of
legumes (86), and as soluble extracellular proteins or
glycoproteins (13,22).
The possibility that an inducer is involved with
macroconidium attachment and agglutination is supported by
the results of the agglutin-induction experiment in
Chapter 6. Macroconidia of F. solani f. sp. phaseoli were
incubated in root homogenate for short periods of time,
washed in fresh nutrient solution, and then resuspended in
fresh nutrient solution. The subsequent agglutination of
macroconidia in the fresh nutrient solution supports the
hypothesis that a diffusible inducer was present in the root
homogenate. It is proposed that the inducer was adsorbed or
bound to reactive sites on macroconidia inducing a
persistent spore adhesiveness that was retained by
macroconidia after spores were removed from the root
homogenate. Based on the involvement of the macroconidium
tips in attachment, agglutination, secretion of mucilage,
and accretion of refuse, the reactive site is proposed to be
on or near the tips of macroconidia.
The possible inducer may also function as a stimulatory
signal for spore germination. Macroconidia germinated
within 1-2 hr when incubated in root homogenate or after

171
contact with roots (Chapter 6). In contrast, few
macroconidia germinated within 5 hr when they were incubated
in fresh nutrient solution containing either mannose,
glucose, or sucrose. When germination did occur in the
presence of carbon and nitrogen sources, the process of
germination was not similar to macroconidium germination in
root homogenate or on root surfaces; macroconidia germinated
primarily from lateral walls of intercalary cells in the
presence of glucose, sucrose, and mannose, but they
germinated from spore tips in root homogenate or on root
surfaces (Chapter 6). These results support the hypothesis
that an inducer may be involved in spore germination.
In the current study, differences were observed in
macroconidium cytology, spore agglutination, and quality of
SEM fixation as the age of macroconidia increased. In
addition, macroconidium age has been reported to affect the
sensitivity of macroconidia during the induction of
agglutination in root homogenate (Chapter 6). These effects
support the possibility that if receptive sites for an
inducer exist on spore tips they may change in either
quality or quantity as macroconidia age.
Additional research is required to clarify the role of
the spore mucilage in macroconidium attachment and
agglutination. Ultrastructure studies would be useful in
determining whether a rapidly secreted thin-layer of
adhesive material is present on spore tips early in the
attachment process. Furthermore, histochemical staining and

172
competitive inhibition studies would help characterize the
chemical nature of the binding phenomenon. In a separate
study (Chapter 6), 20 carbon sources failed to block
agglutination of macroconidia of F. solani f. sp. phaseoli
in a root homogenate of V. radiata. and eight hapten sugars
of plant lectins failed to block attachment of macroconidia
to plant roots. The presence of the proposed inducer should
be confirmed and its mode of action characterized before the
relationship between spore attachment and spore mucilage can
be determined.

CHAPTER 6
EFFECTS OF CARBON SOURCE ON SPORE ATTACHMENT AND GERMINATION
OF MACROCONIDIA OF FUSARIUM SOLANI F. SP. PHASEOLI ON ROOTS
OF VIGNA RADIATA IN HYDROPONIC NUTRIENT SOLUTION
Introduction
Lectins were defined by Goldstein et al. (78) as sugar¬
binding proteins or glycoproteins of nonimmune origin, which
agglutinate cells or precipitate glycoconjugates. Lectins,
also referred to as phytoagglutinins, have been found in a
diversity of plant species encompassing several families of
flowering and nonflowering plants (64). More than 200
species of Phaseolus have been found to contain materials
with hemagglutinating activity (128). Lectins may be
involved in recognition and attachment of nitrogen-fixing
bacteria to legume roots (128,129); they may serve to
protect plants against fungal attack by inhibiting fungal
growth (10,152), or by inhibiting fungal polysaccharases
(128); they may mediate fungal attachment to plant surfaces
(73,91,130); they may be involved in mycoparasitism of
fungal pathogens (9,60,163); and they may mediate attachment
of predatory fungi to nematodes (164). Phytoagglutinins
have been described in seeds (71,179), in leaves and stems
(22), in hypocotyl cell walls (40,86), cotyledons (40), on
root mucilage (80,98), on root hairs (54,74,75,206), and as
extracellular plant proteins or glycoproteins (13,22).
173

174
These and other qualities of plant lectins have been
reviewed (13,64,128,129,163,188) .
A phytoagglutinin has been isolated from hypocotyl cell
walls (86,114,115), seeds (90), and leaves (105) of Viana
radiata (L.) Wilczek. The phytoagglutinin appeared to be a
carbohydrate-binding protein (90,114) that had specific
enzymatic activity (a-galactosidase) (86,90) and could
agglutinate rabbit erythrocytes (90,114). Agglutination was
inhibited by D-galactose and required Ca2+ or Mn2+ ions for
binding activity (114). In addition, the activity of the
phytoagglutinin from V. radiata was shown to be sensitive to
changes in hydrogen ion concentration (114,115). Although
no literature was found that described phytoagglutinins from
roots of V. radiata. the presence of phytoagglutinins in
roots of other legumes (22,54,74,75,206) suggests that they
may be present.
In general, fungal cell walls contain between 80-90%
polysaccharides, with the remainder mostly composed of
proteins and lipids (14). Although nearly a dozen
monosaccharides have been reported in fungal cell walls,
only D-glucose, N-acetylglucosamine, and D-mannose are
consistently found in most fungi (14). Enzymatic hydrolysis
of hyphal cell walls of Fusarium solani (Mart.) Appel. & Wr.
f. sp. phaseoli (Burk.) Snyd. & Hans, yielded 47%
N-acetylglucosamine, 14% glucose, and 6% of an insoluble
residue which contained mannose, galactose, and a uronic
acid (201); the major polysaccharides were identified as

175
chitin and a glucan with B-(l-»3) linkages (201). The chitin
fraction has been shown to probably exist as a core
structure in the conidium walls of F. solani f. sp. phaseoli
(47,118) that is protected by a glucan polymer composed of
a-D-mannopyranosyl residues (118).
Untreated macroconidia of F. solani f. sp. phaseoli
were agglutinated by the lectins concanavalin A and Helix
pomatia L. agglutinin (47,118), indicating that
a-D-mannosyl, a-D-glucosyl, and N-acetyl-a-D-galactosaminyl
residues were likely exposed on the outer surfaces of the
mucilaginous layer of the macroconidium walls.
Consequently, agglutination was blocked by the addition of
the hapten sugars D-glucose, D-mannose, and
N-acetyl-D-galactosamine (47).
In previous studies macroconidia of F. solani f. sp.
phaseoli were shown to attach preferentially to root hairs
of V. radiata (Chapter 4), and were shown to be agglutinated
by a crude extract from roots of V. radiata (Chapter 5).
Based on these results, and on the literature discussed
above, it was hypothesized that a lectin might be involved
in spore attachment by the pathogen F. solani f. sp.
phaseoli to roots of the legume V. radiata. Therefore, the
first objective of this study was to determine if specific
hapten sugars of previously reported plant lectins
(22,54,64,90,128,129,153,188) could block spore attachment
or agglutination by macroconidia of F. solani f. sp.
phaseoli. Inhibition of spore attachment by a specific

176
hapten sugar would indicate that constitutive properties of
both the fungus and the host were involved in attachment and
agglutination. An alternative hypothesis was that
constitutive properties were not involved in spore
attachment, but rather that the macroconidia became adhesive
after they were incubated in a nutrient solution containing
both a carbon and nitrogen source. In support of this
hypothesis, chlamydospores of F. solani f. sp. phaseoli have
been shown to require an exogenous source of carbon and
nitrogen for germination (44), and Jones and Epstein
(112,113) have demonstrated that the adhesion of Nectria
haematococca Berk. & Br. (anamorph: F. solani f. sp.
cucurbitae Snyd. & Hans.) to surfaces was an inductive
process that required both respiration and protein synthesis
in macroconidia. Therefore, the second objective of this
study was to determine the effects of different carbon
sources on the germination and agglutination of macroconidia
of F. solani f. sp. phaseoli.
Materials and Methods
Macroconidia of F. solani f. sp. phaseoli (isolate
F-28A, obtained from A. J. Anderson, Utah State University,
Logan, Utah) were prepared for the spore agglutination and
spore attachment tests using the following procedure.
Macroconidia from modified carnation leaf agar (MCLA)
cultures (Chapter 2) were collected in sterile deionized
water (SDIW). Macroconidia were washed three times by first

177
collecting them on 5.0-jum, cellulose nitrate filters (MSI
Micron Separations, Inc., Westboro, MA, 01581) and then
resuspending them in SDIW. Macroconidia were added to
various treatments and the densities adjusted to 5-10 x 103
spores/ml. A hemacytometer was used to estimate the numbers
of spores in the treatments in this and all subsequent
experiments
The preparation of root homogenate and root leachate
solutions was described previously (Chapter 5). Fresh
nutrient solutions were prepared for each experiment using
the procedures described in Chapter 2. All chemicals were
obtained from Sigma Chemical Co. (St. Louis, MO, 63178),
except D-cellobiose and D-melibiose, which were obtained
from Aldrich Chemical Company, Inc. (Milwaukee, WI, 53233).
All treatments were titrated to pH 5.5 using 0.01 N HNO3
and 0.02 N KOH.
Agglutination tests were conducted at room temperature
(approximately 23-25 C) in separate 50-ml volumes of the
specific treatments. Spore suspensions were gently agitated
using rotary shakers or magnetic stirrers. Incubation
periods differed between experiments and are described
below. After incubation, two, 100-jil samples were withdrawn
from each solution and mounted on acid-washed glass slides.
Samples were viewed under bright field microscopy to
determine if agglutination of macroconidia had occurred.
Spore agglutination was considered positive when at least
four aggregates were observed in a 100-/il sample; each

178
aggregate was composed of at least four individual
macroconidia, in which spore-to-spore contact occurred
between terminal or foot cells of macroconidia.
Twenty carbon sources were tested for their ability to
agglutinate macroconidia of F. solani f. sp. phaseoli in
fresh nutrient solution. Ten- to twelve-day-old
macroconidia were incubated for 1.5 hr in 50-mM solutions of
each of the following carbon sources: a-methyl-D-glucoside,
N-acetyl-D-galactosamine, N-acetyl-D-glucosamine,
N-acetylneuraminic acid, L-arabinose, D-asparagine,
D-cellobiose, D-fructose, D-fucose, L-fucose, D-galactose,
D-gluconic acid, D-glucose, lactose, maltose, D-mannose,
D-melibiose, methyl-a-D-mannopyranoside, L-rhamnose, and
sucrose. Separate 50-ml samples of root homogenate, root
leachate, and fresh nutrient solution were included in each
test as the controls. The experiment was conducted four
times, except for N-acetylneuraminic acid which was tested
twice.
To determine if the various carbon sources would
inhibit spore agglutination in root homogenate, macroconidia
from 10- to 12-day-old MCLA cultures were collected and
washed in SDIW, as was described above. Macroconidia were
then added to separate 50-ml samples of root homogenate,
each of which contained 1 of 20 specific carbon sources
(Table 6-1). Macroconidia were incubated for 1.5 hr in 50-
and 100-mM solutions of each carbon source and viewed under
bright field microscopy, as was described above. Tests with

179
Table 6-1. Effects of carbon source on the agglutination of
macroconidia of Fusarium solani f. sp. phaseoli in nutrient
solution and root homogenate.
Agglutination in
Nutrient solution
with
carbon sources at
Root
carbon
homogenate
with
sources at
Carbon source
50 mM
50 mM
100 mM
a-methyl-D-
glucoside
_z
+
+
D-asparagine
-
+
+
D-cellobiose
-
+
+
D-fructose
-
+
+
D-fucose
-
+
+
D-galactose
-
V
+
D-gluconic acid
-
+
+
D-glucose
-
+
+
D-mannose
-
+
+
D-melibiose
-
+
+
L-arabinose
-
+
+
L-fucose
-
+
+
L-rhamnose
-
+
+
lactose
-
+
+
maltose
-
+
+
methyl-a-D-
mannopyranoside
_
+
+
N-acetyl-D-
galactosamine
_
+
+
N-acetyl-D-
glucosamine
+
+
N-acetylneuraminic
acid
+
+
sucrose
—
+
+
CONTROLS
root homogenate
+
+
+
root leachate
-
V
V
nutrient solution
—
—
—
z - = spore-tip to spore-tip agglutination of macroconidia
was not observed; + = small aggregates of between 5 and 25
macroconidia were observed; and V = variable reaction
(negative and weakly positive responses were observed in
separate tests).

180
50-mM solutions were conducted three times, while tests with
100-mM solutions were conducted twice.
The possibility was considered that a high concentra¬
tion of an agglutinating factor or factors in the root
homogenate might have interfered with the inhibition of
spore agglutination by the different carbon sources; if this
occurred spore agglutination could have been observed even
in the presence of a competitive hapten. A procedure was
developed whereby macroconidia were stimulated to begin
agglutination, but before large aggregates of macroconidia
formed they were removed from the root homogenate and
transferred to solutions of potentially competitive hapten
sugars. The following procedure was used to stimulate the
agglutination of macroconidia. Macroconidia from 10-, 14-,
and 21-day-old MCLA cultures were prepared (hereafter termed
10-, 14-, or 21-day-old macroconidia), as was described
above, and then separately placed in 50-ml samples of root
homogenate for 1, 5, 10, 15, and 20 min. Following
incubation in the root homogenate, macroconidia were
collected on 5.0-^im, cellulose nitrate filters and washed
once by resuspending each sample in 50 ml of fresh nutrient
solution. Macroconidia were again collected on 5.0-nm
filters and then resuspended in 50 ml of fresh nutrient
solution. Macroconidia were incubated in fresh nutrient
solution for 1 hr, and then two, 100-/xl subsamples of the
spore suspensions were viewed under bright field microscopy.
The experiment was conducted three times.

181
Eight hapten sugars were selected to test their ability
to block spore agglutination after macroconidia were
preincubated in root homogenate. Macroconidia from 21-day-
old MCLA cultures were incubated for 20 min in 50 ml of
eight separate samples of root homogenate. Macroconidia
from each sample were collected separately on 5.0-/¿m,
cellulose nitrate filters, and then washed once with 50 ml
of fresh nutrient solution. Macroconidia were then
resuspended in separate 50-ml samples of fresh nutrient
solution, each of which contained one of the following
sugars at 50 mM: D-fucose, D-galactose, D-glucose,
D-mannose, N-acetylneuraminic acid, N-acetyl-D-galactos-
amine, N-acetyl-D-glucosamine, and sucrose. Macroconidia
were agitated gently for 1 hr, and then two, 100-/xl
subsamples of the spore suspensions were viewed under bright
field microscopy. Controls included macroconidia incubated
for 1 hr in 50 ml of each of the following solutions: 1)
freshly prepared root homogenate, 2) fresh nutrient
solution, and 3) fresh nutrient solution after pretreating
macroconidia for 20 min in root homogenate. The experiment
was conducted three times.
To determine if the hapten sugars used in the previous
experiment would block spore attachment to root surfaces,
single, 7- to 8-day-old seedlings of V. radiata were placed
in 200 ml of 50- and 100-mM solutions of D-fucose,
D-galactose, D-glucose, D-mannose, N-acetylneuraminic acid,
N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and

182
sucrose. Macroconidia were collected from 10- to 12-day-old
MCLA cultures and washed three times using the filtration
procedure described above. Plants were preincubated for 15
min in each sugar solution, and then inoculum was added to
each treatment so that the final spore densities were 4-5 x
103 propagules/ml. Inoculated plants were gently agitated
for 10 min on a rotary shaker. Plants inoculated in fresh
nutrient solution, without the hapten sugars, served as
controls. At the end of the inoculation period, roots were
severed at the crown of each plant and stained for 5 min in
0.05% trypan blue in a lactic acid:water:glycerol (1:1:2)
solvent (Chapter 2). Second-order roots (sensu Fitter (70))
with root hairs were mounted on glass slides and viewed with
bright field microscopy. The experiment was conducted twice
for each sugar concentration.
To determine if macroconidium germination in specific
sugar solutions was similar to spore germination in root
homogenate, or to spore germination on root surfaces of
V. radiata. macroconidia of F. solani f. sp. phaseoli were
incubated in separate 50-mM samples of D-fucose,
D-galactose, D-glucose, D-mannose, N-acetylneuraminic acid,
N-acetyl-D-galactosamine, N-acetyl-D-glucosamine, and
sucrose. Sugars were dissolved in separate 50-ml volumes of
fresh nutrient solution. Three additional treatments were
tested that included different combinations of sugars. In
one treatment a mixture of the eight sugars was prepared in
a single volume of 50 ml of fresh nutrient solution so that

183
the final concentration of each sugar was 10 mM. To
determine if galactose inhibited the germination of apical
cells of macroconidia, D-galactose was combined with
D-glucose and then separately with sucrose so that each
sugar was at a concentration of 50 mM in each of the two
50-ml samples. Control treatments consisted of 50 ml each
of fresh nutrient solution and root homogenate.
Macroconidia were collected from 10- to 12-day-old MCLA
cultures and washed three times in SDIW, as was described
above. Macroconidia were added to each solution, and the
solutions incubated for 24 hr with continuous agitation at
room temperature. Two, 100-/il samples were withdrawn from
each solution at 1.5, 5, and 24 hr; samples were mounted on
acid-washed glass slides and then viewed under bright field
microscopy. After 24 hr, macroconidia were collected onto
Whatman No. 42 filter paper using vacuum filtration.
Without permitting the desiccation of macroconidia, discs of
filter paper were transferred to fresh nutrient solution and
cut into 4.0-mm2 pieces. Sections of filter-paper from each
treatment were prepared for scanning electron microscopy
(SEM) by fixation in 1.0% unbuffered osmium tetroxide
containing 0.003% Photo-flo (Eastman Kodak Company,
Rochester, NY, 14650), as was described previously
(Chapter 5). Samples were photographed with Polaroid® 4x5
Instant Film, Type N55 (Polaroid Corp., Cambridge, MA,
02139) using a Hitachi S-530 scanning electron microscope
(Hitachi Instruments, Inc., Danbury, CT, 06810). Tests

in which spore germination was studied using bright field
microscopy were conducted four times, while tests using SEM
were conducted twice.
Results
Spore-tip to spore-tip agglutination of macroconidia of
F. solani f. sp. phaseoli was not observed in nutrient
solution treatments containing 50-mM concentrations of the
various carbon sources (Table 6-1). Conversely, spore-tip
to spore-tip agglutination was observed in all 50- and
100-mM treatments of root homogenate plus the various carbon
sources. Root homogenate and nutrient solution control
treatments responded consistently throughout these tests;
agglutination occurred in root homogenate, but was always
absent in nutrient solution treatments. In contrast,
agglutination of macroconidia in root leachate was variable;
either negative or weakly positive results were observed in
separate repetitions of each test. Acid-washed glass slides
were essential for determining spore agglutination because
macroconidia attached to refuse present on uncleaned slides
and formed aggregates of spores which appeared similar to
agglutinated spores in root homogenate.
Induction of spore agglutination did not occur with 10-
or 14-day-old macroconidia, but induced spore agglutination
was observed with 21-day-old macroconidia (Table 6-2). The
induced response was observed with 21-day-old macroconidia
incubated in root homogenate for 1-20 min before

185
Table 6-2. Effects of macroconidium age on the agglutin¬
ation of spores of Fusarium solani f. sp. phaseoli in
nutrient solution after incubation of macroconidia in root
homogenate.
Time in
root
homogenate
(min)x
Agglutination
of macroconidia
10
days^ 14
days
21
days
0 hr
1 hr 0 hr
1 hr
0 hr
1 hr
1
_z
-
-
-
+
5
-
V
-
-
+
10
V
V
-
-
+
15
V
+w
-
V
++
20
+w
+w
V
+w
++
CONTROLS
NS
-
-
-
-
-
RH
-
+
+
-
++
x Macroconidia
were
incubated in root
homogenate for ]
L-20
min and then transferred to fresh nutrient solution and
incubated for an additional 1 hr. For the controls
macroconidia were incubated in fresh nutrient solution
(NS) and root homogenate (RH) for 1 hour.
Â¥ Spore agglutination was determined at 0 and 1 hr after
macroconidia were transferred from root homogenate to
fresh nutrient solution. Macroconidia were from 10-, 14-,
and 21-day-old modified carnation leaf agar cultures.
z - = spore-tip to spore-tip agglutination of macroconidia
was not observed; +W = weak positive reaction; + = small
aggregates of between 5 and 25 macroconidia were observed;
++ = large aggregates of greater than 100 macroconidia
were observed; and V = variable reaction (negative and
weakly positive responses were observed in separate
tests).

186
transferring spores to fresh nutrient solution. Weak
positive reactions were observed with 10- and 14-day-old
macroconidia incubated for 20 min in root homogenate, but
aggregates broke up and dispersed during the 1 hr incubation
period in fresh nutrient solution. Reactions were
inconsistent with 10- and 14-day-old macroconidia incubated
for 5-15 min in root homogenate; both weakly positive and
negative reactions were observed among tests, but when
aggregates were formed they were not maintained during
subsequent incubation in fresh nutrient solution.
None of the eight hapten sugars inhibited spore-tip to
spore-tip agglutination when macroconidia were stimulated to
agglutinate in root homogenate (Table 6-3). In addition,
the eight hapten sugars did not inhibit macroconidium
attachment to root surfaces of V. radiata in fresh nutrient
solution at either 50- or 100-mM concentrations of the
sugars. Macroconidia attached to roots equally well in all
treatments, including the fresh nutrient solution
treatments, which were free of any added sugars.
Macroconidia germinated within 1.5 hr when incubated in root
homogenate or when they were attached to root surfaces of V.
radiata. Germ tubes were not observed on macroconidia
incubated in the hapten sugars for 1.5 hr. Germ tubes were
observed after 5 hr from a very low number of macroconidia
incubated in D-glucose, D-mannose, and sucrose. Germ tubes
were observed at 24 hr in all treatments except D-fucose,
D-galactose, and fresh nutrient solution samples.

187
Table 6-3. Effects of hapten sugars on spore agglutination
and attachment of macroconidia of Fusarium solani f
ohaseoli to roots of Vicrna radiata.
. sp.
Spore agglutination in
Spore attachment to
sugar solutions
after
roots of
Vicrna
20 min in root
radiata incubated in
homogenate at
time
sugar solutions at
Sugars
0 hr
1 hr
50 mM
100 mM
D-fucose
_y
+
++
++
D-galactose
-
+
++
++
D-glucose
-
+
++
++
D-mannose
-
+
++
++
N-acetylneur-
aminic acid
+
++
++
N-acetyl-D-
galactosamine
+
++
++
N-acetyl-D-
glucosamine
—
+
++
++
sucrose
—
+
++
++
CONTROLS2
NS
++
++
RH - 20 min
-
+
NT
NT
RH - 1 hr
-
++
NT
NT
y - = agglutination or attachment of macroconidia from 21-
day-old cultures to root surfaces were not observed; + =
spore agglutination or spore attachment to root surfaces
were observed; ++ = large aggregates of greater than 100
macroconidia were observed in sugar solutions or large
numbers of macroconidia attached to roots; and NT =
treatment not tested.
z Control treatments were one or more of the following:
fresh nutrient solution (NS) without hapten sugars;
macroconidia were incubated 20 min in root homogenate, and
then for 1 hr in fresh nutrient solution (RH - 20 min);
and macroconidia were incubated for 1 hour in root
homogenate (RH - 1 hr).

188
Germ tubes emerged from lateral walls of intercalary cells
when macroconidia were incubated in hapten sugars (Figure
6-1A), but they emerged primarily from terminal and foot
cells when macroconidia were incubated in root homogenate
(Figure 6-1B). In addition, macroconidia primarily
germinated from terminal and foot cells when attached to
root surfaces.
An amorphous material, termed spore mucilage
(Chapter 5), was associated with germ tube emergence from
terminal and foot cells when macroconidia were incubated in
root homogenate (Figure 6-1B). Spore mucilage was observed
at the sites of germ tube emergence at spore tips, but was
not observed on the elongating germ tubes. Spore mucilage
was not observed when germ tubes emerged from lateral walls
of intercalary cells of macroconidia incubated in hapten
sugars (Figure 6-1C and D); germ tubes appeared to
physically break-through lateral walls of intercalary cells
leaving a ragged-edged tear in the macroconidium walls
(Figure 6-1C and D).
Very few macroconidia germinated in solutions of
D-fucose, D-galactose, or fresh nutrient solution without
hapten sugars after 24 hr (Figure 6-2A). However, most
macroconidia germinated in solutions of D-mannose,
D-glucose, sucrose, and root homogenate controls.
Macroconidia incubated in D-glucose, D-mannose, and sucrose
exhibited severely swollen intercalary cells (Figures 6-2B
and C). The few macroconidia that did germinate often

Figure 6-1. Macroconidia of Fusarium solani f. sp. phaseoli
observed in the solutions of hapten sugars and root
homogenate. A, Germ tubes (gt) emerged from intercalary
cells of macroconidia (ma) incubated for 5 hr in 50-mM
solutions of D-glucose, D-mannose, and sucrose.
B, Germ tubes emerged from terminal and foot cells of
macroconidia incubated for 1.5 hr in root homogenate; a
spore mucilage (sm) was observed at the sites of germ tube
emergence. C,D, Spore mucilage was not observed when germ
tubes emerged from intercalary cells of macroconidia that
were incubated in hapten sugars; germ tubes appeared to
mechanically rupture macroconidium walls during germination
(arrows).

06T

Figure 6-2. Macroconidia of Fusarium solani f. sp. phaseoli
observed after 24 hr in 50-mM solutions of hapten sugars.
A, Few macroconidia germinated in solutions of D-fucose and
D-galactose, nor did they germinate in fresh nutrient
solution. B,C, Swollen intercalary cells (si) were observed
in macroconidia (ma) incubated in D-glucose, D-mannose, and
sucrose; conidiophores (co) and secondary macroconidia (sma)
developed directly from intercalary cells, but not terminal
cells (tc), of macroconidia. D, A material similar to spore
mucilage was observed on germ tubes (gt) and conidiophores
of macroconidia incubated with D-glucose, D-mannose, and
sucrose; however, the material did not accumulate at the
points of germ tube emergence (arrows), as was observed in
root homogenate.

361

193
immediately produced conidiophores and secondary macro-
conidia (Figures 6-2B and C). The immediate production of
secondary macroconidia from germinating spores of F. solani
f. sp. phaseoli was not observed when macroconidia were
incubated in root homogenate, nor when macroconidia were
attached to root surfaces. The surfaces of germ tubes and
conidiophores of macroconidia incubated in the hapten sugars
appeared to be covered with a material similar to spore
mucilage (Figures 6-2C and D), but the origin of this
material was unclear.
Macroconidia incubated in the mixtures of hapten sugars
germinated in a manner consistent with the processes
observed with D-glucose, D-mannose, and sucrose, except
germination appeared to progress more rapidly. A greater
number of spores incubated in the solutions of sugars
germinated by 24 hr than in solutions of the individual
sugars. Macroconidia incubated in combinations of
D-galactose plus sucrose and D-galactose plus D-glucose
germinated in a manner consistent with macroconidium
germination observed in nutrient solution samples containing
either sucrose or D-glucose.
Discussion
The hypothesis that a root lectin might be involved in
spore attachment by macroconidia of F. solani f. sp.
phaseoli to roots of V. radiata was not supported by the
results from the current study. In one experiment, 20

194
different carbon sources, including eight hapten sugars of
previously described plant lectins (22,54,64,90,128,129,
153,188), failed to block spore agglutination at either 50-
or 100-mM concentrations when macroconidia were incubated in
root homogenate. Using a different procedure, in which
macroconidia were induced to become adhesive, the eight
hapten sugars again failed to block spore agglutination.
And in a third experiment, the eight hapten sugars failed to
block attachment of macroconidia to roots of V. radiata.
Furthermore, the phytoagglutinin from V. radiata was shown
by Kauss and coworkers (114,115) to be sensitive to [H+];
hemagglutinating activity of the lectin was greatest at
pH 7.0-8.0, and dropped dramatically when pH was less than
7.0. The effects of [H+] on the activity of the V. radiata
lectin (114,115) are not in agreement with previous studies
(Chapters 4 and 5), which showed that spore attachment to
roots and spore agglutination in root homogenate were
suppressed at pH 7.0 and increased as pH was lowered to 4.0.
Based on these results, the phytoagglutinin previously
reported for V. radiata (86,90,114,115,138) does not appear
to be involved in attachment of macroconidia of F. solani f.
sp. phaseoli to roots.
Although the mechanism that mediates spore attachment
in the pathosystem of F. solani f. sp. phaseoli and V.
radiata is not known, adsorptive- and protein-mediated
mechanisms may be involved (Chapter 4). This conclusion was
based on the effects that temperature and [H+] had on spore

195
attachment to roots (Chapter 4). In Chapter 5, an inducer
of plant origin was proposed as the factor involved in both
the stimulation of spore germination and in the induction of
spore adhesiveness. The results from the agglutin-induction
experiment in the current study are consistent with the
conclusions discussed in Chapters 4 and 5. In the current
study, 21-day-old macroconidia became adhesive after only 1
min in root homogenate.
The proposed inducer may be a structural component of
the root surface or it may be a soluble component of the
root apoplast or symplast. In a preliminary test, a root
leachate solution was prepared by severing roots of V.
radiata at the crowns and incubating them in fresh nutrient
solution for 18 hr; a strong spore agglutination response
was observed in this solution. Subsequently, the procedure
was modified so that only intact plants were used for
preparing root leachate. The result of this preliminary
test indicates that symplastic fluids of V. radiata roots
may contain an inducer. In addition, the inconsistent
agglutination response with the different batches of root
leachate supports the conclusion that plant roots may be
wounded during the preparation of root leachate, even though
seedlings of V. radiata were produced in specially designed
trays, as was described in Chapter 2, to minimize root
damage. Caution should be exercised when root leachates are
used in phytoagglutinin studies because the leachate may

contain symplastic fluids that normally would not be
secreted by the roots.
196
Carbon and nitrogen sources appear to be required for
the germination of macroconidia and chlamydospores of
several Fusarium spp. (44,113,136,143). In addition, carbon
and nitrogen sources may be required for the attachment of
macroconidia of N. haematococca to plant surfaces (113).
However, in the current study spore germination in the
different carbon sources differed greatly from spore
germination in root homogenate or on roots of V. radiata.
Macroconidia germinated within 1.5 hr when incubated in root
homogenate, but required at least 5 hr to germinate in
nutrient solutions containing either D-glucose, D-mannose,
or sucrose. Very few of the macroconidia incubated in
nutrient solution containing D-fucose and D-galactose
germinated within 24 hr. Macroconidia of F. solani f. sp.
phaseoli germinated within 2-3 hr when attached to root
surfaces under conducive conditions of temperature and [H+]
(Chapter 4). Germination of macroconidia of F. culmorum
(Smith) Sacc. required 4-6 hr when spores were incubated in
distilled water containing glucose and a nitrogen source
(136). Furthermore, Griffin (81) reported that most
macroconidia of F. solani f. sp. phaseoli incubated in
distilled water containing glucose and KNO3 germinated after
12 hr.
Three additional anomolies were observed with
macroconidium germination in D-glucose, D-mannose, and

197
sucrose solutions. First, severely swollen intercalary
cells of macroconidia were consistently observed when spores
were incubated in the sugar solutions. Swollen intercalary
cells were not observed when macroconidia were incubated in
root homogenate, nor were they observed when spores
germinated on root surfaces (Chapter 4). Marchant and White
(136) described a similar swelling of intercalary cells of
macroconidia of F. culmorum when spores were incubated in
distilled water containing glucose and a nitrogen source.
Second, germ tubes emerged primarily from intercalary cells
of macroconidia when spores were incubated in the sugar
solutions. In contrast, germ tubes emerged primarily from
spore tips when macroconidia were incubated in root
homogenate, or when they were allowed to germinate on root
surfaces. Receptive sites on macroconidium tips have been
proposed for an inducer that might function in both spore
attachment and in spore germination (Chapter 5). And third,
conidiophores and secondary macroconidia developed directly
from germinating macroconidia when spores were incubated in
nutrient solution containing D-glucose, D-mannose, or
sucrose. This response was not observed in any treatments
in which macroconidia germinated on the root surface, nor
was the response observed when macroconidia germinated in
root homogenate. These results are not in agreement with
the principle that fungal sporulation occurs when the
available food base is depleted (134); the sugars appeared
to stimulate the production of secondary macroconidia.

198
Furthermore, these results may provide an explanation for
why the addition of sucrose to a nutrient solution 2 days
before plant inoculation increased the severity of disease
in wheat caused by Helminthosporium sativum P. K. & B. (23);
the addition of sucrose may have increased the inoculum
density of the pathogen in the nutrient solution.
These results support the conclusion that a factor
other than the simple availability of carbon and nitrogen is
involved with spore attachment and germination on plant
roots in a hydroponic nutrient solution. The chemical
nature of the proposed inducer is not known. It is possible
that a specific carbon source, other than those tested in
the current study, might mediate spore attachment and
germination on root surfaces. Alternatively, a specific
combination of individual carbon sources might be required
by macroconidia to germinate in a manner similar to the
in situ process observed in root homogenate or on root
surfaces. However, in the current study three combinations
of sugars failed to induce normal spore germination;
macroconidia germinated in the combinations of sugars in a
manner consistent with spore germination in nutrient
solutions separately containing D-glucose, D-mannose, and
sucrose.

CHAPTER 7
SUMMARY AND CONCLUSIONS
Microbial contamination of controlled ecological life
support systems (CELSS) will likely include fungi that are
ecologically adapted to survival on plants originally grown
in terrestrial environments. Although gnotobiotic plant
production systems have been proposed for CELSS (154), it
seems unlikely that the extreme amount of effort required to
maintain a gnotobiotic system (122,154) will justify their
development. Similar species of fungi have been reported in
American spacecraft (24,96,144,180,181,218), ground-based
CELSS modules (94,127), and a hydroponic system (174).
Fusarium spp. were selected for the current study based on
their recovery from these closed systems (94,96,127,144,174
180,181), and because they comprise the second largest group
of root pathogens in terrestrial hydroponic systems
(45,106,165,174,176,177,182,183,184,212,226).
Species of Acremonium. Aspergillus. Cephalosporium.
Chaetomium. Cladosporium. Curvularia. Cvlindrocarpon.
Fusarium. Mucor. Penicillium. Stachvbotrvs. and Trichoderma
were isolated from the nutrient solution in both inoculated
and noninoculated treatments in the experiments on the
effects of temperature, hydrogen ion concentration ([H+]),
humidity, and light quality on disease (Chapter 3). These
199

200
fungi were usually recovered from the nutrient solution at
low densities, but species of Cladosporium were recovered
from treatments at densities as high as 200 propagules/ml.
Carbon-based materials in contact with the nutrient solution
were believed to be the source of fungal contamination.
Researchers using hydroponic systems should consider the
stability of the materials used in the systems and the
effects of these materials on microorganisms. Microbial
fouling of hydroponic systems might result in 1) the
clogging of tubing, 2) the degradation of electrical
sensors, 3) the induction of allelopathic or toxigenic
effects, or 4) the interference with desired microbiological
processes.
Root necrosis was observed on most cultivars of
Phaseolus vulgaris L., Pisum sativum L., Triticum
avestivum L., and Viqna radiata (L.) Wilczek when plants
were inoculated with various Fusarium spp. However, symptom
development was highly variable and appeared to be dependent
on inoculum density. Plant diseases caused by Fusarium spp.
in terrestrial hydroponic systems have been reported as
severe (45,84,165,182) or as minimal (51,66,177,184,212).
The variability in plant disease caused by Fusarium spp. in
hydroponic systems might be due to differences among the
pathosystems in 1) the resistance of the host or the
virulence of the pathogen, 2) the temperature and [H+]
optima for disease development, or 3) the ability of the
infective propagules to attach to plant roots. In the

201
current study, the latter two possibilities were shown to
affect plant disease caused by F. solani f. sp. phaseoli in
V. radiata.
Of the environmental parameters studied, temperature
appeared to impart the greatest effect on disease caused by
F. solani f. sp. phaseoli. Disease was greatest at 20 C and
declined steadily until at 30 C plant weight differences
were not observed between noninoculated and inoculated
plants. The effects of temperature on disease were similar
to those on other bean cultivars infected with F. solani f.
sp. phaseoli (33,146,147). In addition, temperature
affected the production of secondary inoculum on roots of V.
radiata. Macroconidia of the pathogen were produced on
sporodochia, highly branched monophialides, and individual
conidiophores. The numbers of conidiophores observed on the
root surface, and the numbers of propagules of the pathogen
recovered from the nutrient solution, decreased as
temperature increased.
The effects of [H+] on the severity of disease were
tested in two separate experiments in which the timing of
the [H+] treatments differed. In the first study (Chapter
3), the pH for all treatments was maintained at 6.0 during
plant inoculation and then readjusted in specific treatments
to pH 4.0, 5.0, 6.0, and 7.0 after 24 hr. In the second
study (Chapter 4), various [H+] were maintained for 24 hr
after inoculation and then readjusted to pH 6.0. In the
first study, the post-attachment and post-infection

202
processes of pathogenesis were influenced by [H+], where as
in the second experiment spore attachment and host infection
processes of pathogenesis were influenced by the [H+]. In
the first experiment the severity of disease was suppressed
at pH 4.0 compared to pH 5.0, 6.0, and 7.0, but in the
second experiment the severity of disease was suppressed at
pH 7.0 compared to pH 4.0, 5.0, and 6.0. The effects of
[H+] on disease in the second experiment correlated with the
effects of [H+] on spore attachment; the severity of disease
was greatest and spore attachment to roots was highest at
pH 4.0. These results support the hypothesis that [H+]
affects pathogenesis differently depending on the timing of
the [H+] treatment. If confirmed, [H+] may be a useful and
flexible control method for managing root diseases caused by
F. solani f. sp. phaseoli in hydroponic systems. Altering
the [H+] of the nutrient solution has been reported to
affect disease caused by Phvtophthora cinnamomi Rands (19)
on avocado and Pvthium debaryanum Hesse on lettuce (236).
Clearly, the effects of [H+] on spore attachment and disease
in hydroponic systems should be studied with other root
pathogens.
Spore attachment to root surfaces, agglutination of
macroconidia in root homogenate, secretion of spore mucilage
during germination, and accretion of refuse to spore tips
were similarly affected by changes in temperature and [H+].
The processes were all suppressed at pH 3.0 and 7.0, and at
35 C. Based on the these responses, it was proposed that an

203
inducer may be present in roots of V. radiata that might be
involved in spore attachment, agglutination, and
germination. It was hypothesized that the inducer might be
a plant lectin (phytoagglutinin), and research was initiated
to determine if the attachment process could be inhibited by
the addition of hapten sugars. If inhibition could be
induced, the chemical nature of the reactive site might be
inferred, and a possible method for suppressing spore
attachment to roots might be discerned.
A phytoagglutinin has been isolated from hypocotyl cell
walls (86,114,115), seeds (90), and leaves (105) of
V. radiata. Based on reports of lectins in roots of other
legumes (22,54,74,75,206), it was concluded that the
phytoagglutinin probably also occurred in roots of V.
radiata. Results from the competitive sugar experiments did
not support the hypothesis that lectins were involved in
spore attachment to roots. Hapten sugars failed to
stimulate spore agglutination in nutrient solution, block
agglutination in root homogenate, or block spore attachment
to roots of V. radiata.
The mechanism of attachment of macroconidia of
F. solani f. sp. phaseoli to roots of V. radiata. and the
chemical nature of the proposed inducer, are not known.
Results from these experiments are consistent with the
hypothesis that a protein or glycoprotein is involved in
spore attachment. If confirmed, protein inhibitors might be
useful for suppressing spore attachment of the pathogen to

204
root surfaces. Jones and Epstein (112,113) have
demonstrated that the adhesion of Nectria haematococca
Berk. & Br. (anamorph: F. solani f. sp. cucurbitae Snyd. &
Hans.) to polystyrene was significantly reduced when
respiration and protein inhibitors were present.
An integrated pest managemnet (IPM) program has been
proposed for space-based CELSS (158,159). Similarities
exist between terrestrial hydroponic systems and plant
production systems proposed for CELSS (30,56,178,166,235),
and it seems likely that an IPM program might be developed
for CELSS by utilizing the information derived from
terrestrial systems. Various methods have been employed to
suppress root pathogens in hydroponic systems (190,191,
210,224), but limitations in efficacy, phytotoxicity,
applicability, and energy reguirements may reduce their
utility in CELSS. Ultraviolet radiation induced severe
plant chlorosis in lettuce (199,210) and tomato (50),
presumably by destroying the chelating agents for Fe, which
resulted in the precipitation of nutrient salts from the
solution (50,199,210). Ozonation (190) may not be usable in
a closed-system like a CELSS because the O3 must be removed
from solution and vented to minimize its biocidal
activities. Ultra-filtration has been reported to be
effective in removing fungi and bacteria from nutrient
solution (190,199). However, an underlying principle of
CELSS is to minimize or eliminate resupply of materials from
Earth, and a filtration system would likely require regular

205
replacement of the filter membranes. Heat treatment has
been reported to be effective for the control of tobacco
mosaic virus and Verticillium dahliae Kleb on tomato in a
recirculating nutrient solution (191). However, energy is a
limiting factor in space flight (124,166), and a heating
system would likely require a great deal of energy to both
heat and then to subsequently cool the nutrient solution.
Strict air and water quality protocols for American
spacecraft (18) appear to preclude the use of chemical
biocides in CELSS.
Finally, bacterization has been effective in delaying
the onset of disease symptoms of plants (4) or in reducing
plant wilt (224) in hydroponic systems. Bacterization may
be ideally suited for disease suppression in hydroponic
systems, and research should be continued in this area.
However, until the effects of microgravity on plant
(46,87,121,155,156,202) and microbial (2,139,170,214,240)
physiology are better understood, bacterization may pose a
threat to a space-based CELSS. It might be unwise to
inoculate a CELSS with theoretically benign microorganisms
until the effects of microgravity on their biology,
pathology, and survival are studied.
Components of CELSS plant production systems will be
engineered for maximum controllability and compartmental-
ization to optimize horticultural conditions for specific
crops (124). Environmental control of each compartment may
offer an effective method for managing microbial

206
interactions with plants. Results from the current study
support the conclusion that temperature and [H+] can alter
the severity of root disease caused by F. solani f. sp.
phaseoli in a hydroponic system. Temperature and [H+] have
been effective in suppressing other diseases in hydroponic
systems (15,19,77,236).
The physical characteristics of a CELSS also may be
important to minimize plant disease. Plant production
systems should be isolated in separate modules in which each
unit includes a mixture of compatible plant species such
that the redundant units each contain a proportion of the
total biomass of each plant species. Thus, if the
productivity of an individual unit is damaged by a
microbiological problem, the loss would not result in the
complete elimination of one species. In addition,
compartmentalization would permit the temporary shut-down of
a unit for the sanitation of microbially contaminated
systems.
The design of individual plant growing subsystems also
may facilitate disease control and increase stability and
reliability of a CELSS. Bugbee and Salisbury (30) advocated
the design of hydroponic systems for CELSS which are not
vunerable to power failures. Plants grown in aeroponic and
NFT systems may be rapidly damaged by mechanical breakdowns
that interrupt the flow of nutrients. Furthermore, the
design of a plant growing system may facilitate the
suppression of plant disease. Rattink (184) reported that

207
the physical characteristics of an ebb and flow hydroponic
system prevented the rapid dispersal of propagules of F.
oxvsporum (Schlecht.) f. sp. cvclaminis Gerlach. Further
research should be conducted on how the physical attributes
of hydroponic systems affect the dynamics of plant disease.
Research into the biology, etiology, and epidemiology
of plant pathogens in terrestrial hydroponic systems should
be applicable to the development of an IPM program for
CELSS. Research topics might include studies on 1) the
ability of spores and mycelium of various potential
pathogens to attach to plant roots in hydroponic systems;
2) the effects of [H+] on spore attachment and disease in
various pathosystems; 3) the effects of [H+] on the biology
and ecology of bacteria in hydroponic systems; 4) the
bacterization and bio-fouling of the hydroponic systems as
they relate to fluctuations in temperature, [H+], and
conductivity of the nutrient solution; 5) the allelopathic
and toxigenic interactions among plants and microorganisms;
and 6) the methods of sanitizing or sterilizing hydroponic
systems.

APPENDIX A
DRY WEIGHTS AND SHOOT-ROOT RATIOS FROM
PATHOGENICITY TESTS

209
Table A-l. Effects of temperature on disease caused by Fusarium solani f. sp. phaseoli in
Viana radiata.
Temperature
(C)
Plant dry
weight (g)
Shoot-root
ratios^
Noninoculated
Inoculated
Noninoculated
Inoculated
20
0.39 az
0.22* a
3.5 a
5.3* a
24
0.68 b
0.60 b
3.9 b
4.1 b
28
0.99 c
0.87 c
4.8 c
4.9 c
32
1.05 d
1.03 d
5.9 d
5.7 d
36
0.80 b
0.84 c
8.2 e
6.9* e
y Shoot-root ratios were estimated using dry weights of plant tissues.
z Table values represent the means of 12 replicates per treatment (four replicates per
treatment in each of three experiments). Data were transformed to logs and then
subjected to an orthogonal polynomial contrast analysis; table values are presented as
detransformed numbers. The temperature effect was highly significant and best fit by a
quadratic polynomial equation (P < 0.01). Least-squares mean separation tests were used
to examine pairwise comparisons between appropriate treatments. Significant differences
in comparisons between inoculated and noninoculated plants at each temperature are
indicated by an asterisk (*) (P < 0.05). Treatments in columns followed by the same
letter were not different (P > 0.05).

Table A-2. Effects of hydrogen ion concentration and temperature on disease caused by
Fusarium
solani f. sp.
phaseoli in Vicrna
radiata.
PH
Temperature
(C)
Plant dry
weight (g)
Shoot-root
ratios^
Noninoculated
Inoculated
Noninoculated
Inoculated
4.0
20
0.40
az
0.21* b
3.8
a
7.0* b
25
0.77
b
0.82 c
5.0
b
5.9 a
5.0
20
0.39
a
0.16* a
3.8
a
7.8* b
25
0.83
b
0.69 c
5.0
b
5.7 a
6.0
20
0.41
a
0.17* ab
3.7
a
7.1* b
25
0.80
b
0.73 c
5.2
b
5.1 a
7.0
20
0.40
a
0.14* a
3.5
a
6.3* b
25
0.71
b
0.68 c
5.2
b
4.7 a
y Shoot-root ratios were estimated using dry weights of plant tissues.
z Table values represent the means of six replicates per treatment (two replicates per
treatment in each of three experiments). Data were adjusted with a 0.25-power
transformation and then subjected to an orthogonal polynomial contrast analysis; table
values are presented as detransformed numbers. The individual effects of temperature
and hydrogen ion concentration were significant and best fit by linear polynomial
equations for both fresh weights and dry weights (P < 0.05). An interactive effect
between temperature and the hydrogen ion concentration was not observed (P > 0.10).
Least-squares mean separation tests were used to examine pairwise comparisons between
appropriate treatments. Significant differences in the comparisons between
noninoculated and inoculated plants for each temperature and hydrogen ion concentration
are indicated by an asterisk (*) (P < 0.05). Treatments in columns followed by the same fo
letter were not different (P > 0.05). 1-1
o

Table A-3. Effects of humidity and temperature on disease caused by Fusarium solani f.
sp. phaseoli on Viana radiata.
Humidity
(mm Hg, VPD)X
Temperature
(C)
Plant dry weight (g)
Shoot-root
ratios^
Noninoculated
Inoculated
Noninoculated
Inoculated
6.0
20
0.43
az
0.23* a
4.0
a
5.8* a
25
0.97
b
0.85 b
5.2
b
5.4 a
30
1.02
b
1.10 c
6.7
c
6.8 b
12.0
20
0.42
a
0.23* a
3.9
a
5.7* a
25
1.04
b
0.88 b
5.0
b
5.5 a
30
1.04
b
1.07 c
5.9
c
6.1 ab
x Humidity was measured as vapor pressure deficit (VPD) in mm Hg; vapor pressure deficits
of 6.0 and 12.0 mm Hg, at 25 C, correspond to 75% and 45% relative humidities,
respectively.
y Shoot-root ratios were estimated using dry weights of plant tissues.
z Table values represent the means of eight replicates per treatment (four replicates per
treatment in each of two experiments). Data were transformed to logs and then subjected
to an orthogonal polynomial contrast analysis; table values are presented as
detransformed numbers. Effects of humidity and of a humidity-by-temperature interaction
were not observed (P > 0.10). The effect of temperature was highly significant and best
fit by a quadratic polynomial equation (P < 0.01). Least-squares mean separation tests
were used to examine pairwise comparisons between appropriate treatments. Significant
differences in the comparisons between noninoculated and inoculated plants at each
temperature and humidity are indicated by an asterisk (*) (P < 0.05). Treatments in
columns followed by the same letter were not different (P > 0.05).

Table A-4. Effects of light quality and temperature on disease caused by Fusarium solani
f. sp. phaseoli in Vigna radiata.
Lamp-type
Temperature
(C)
Plant dry weight (g)
Shoot-root
ratios^
Noninoculated
Inoculated
Noninoculated
Inoculated
Fluorescent
20
0.42
az
0.21*
a
3.8
a
7.0* b
25
0.70
b
0.52*
b
5.3
c
5.0 a
Metal halide
20
0.39
a
0.19*
a
4.0
a
7.6* b
25
0.68
b
0.50*
b
5.1
be
5.4 a
High pressure
20
0.44
a
0.22*
a
4.3
ab
7.5* b
sodium
25
0.86
b
0.66*
b
5.9
c
6.4 a
y Shoot-root ratios were estimated using dry weights of plant tissues.
z Table values represent the means of 12 replicates per treatment (four replicates per
treatment in each of three experiments). Data were transformed to logs and then
subjected to an Analysis of Variance and a least-squares mean separation test; table
values are presented as detransformed numbers. Effects of light quality on disease
development and plant growth, an interaction between temperature and light quality, and
an interaction between inoculum and light quality were not observed (P > 0.10).
Significant effects of temperature and inoculum were observed (P < 0.05). Significant
differences in the comparisons between noninoculated and inoculated treatments at each
temperature and lamp-type are indicated by an asterisk (*) (P < 0.05). Treatments in
columns followed by the same letter were not different (P > 0.05).

213
Table A-5. Effects of temperature on root length of Lactuca
sativa in bioassays from experiments on disease caused by
Fusarium solani f. sp. phaseoli in Viqna radiata.
Temperature
Root length
(mm) y
(C)
Noninoculated
plants
Inoculated
plants
20
25.3Z
23.4
24
25.5
26.2
28
25.8
27.1
32
27.3
26.3
36
23.4
24.7
y Root lengths of 40-45 four-day-old seedlings of L. sativa.
cv. 'Grand Rapids,' were measured using the Microcomp®
Video Image Analysis System.
z Samples from each replicate per treatment, for each
experiment, were bulked and processed as one sample.
Table values represent the means of three bulked samples
(one sample per treatment in each of three experiments).
Data were analyzed using an orthogonal polynomial contrast
analysis. Effects of temperature and inoculation were not
observed (P > 0.10) for root elongation of L. sativa. A
second analysis was conducted to test whether any
treatments differed from non-treatment control bioassays,
which were made using fresh nutrient solutions (the mean
of three replicates was 24.5 ; no effects were observed
(P > 0.10).

214
Table A-6. Effects of hydrogen ion concentration and
temperature on root length of Lactuca sativa in bioassays
from experiments on disease caused by Fusarium solani f. sp.
phaseoli in Viana radiata.
PH
Temperature
(C)
Root length
(mm) ¥
Noninoculated
plants
Inoculated
plants
4.0
20
19.8Z
20.4
25
19.4
18.7
5.0
20
20.3
19.1
25
20.5
19.7
6.0
20
19.7
20.1
25
19.6
20.2
7.0
20
20.9
21.2
25
20.4
19.9
y Root lengths
of 40-45, four-
-day-old seedlings
of L.
sativa. cv. 'Grand Rapids,' were measured using the
Microcomp® Video Image Analysis System.
z Samples from each replicate per treatment, for each
experiment, were bulked and processed as one sample.
Table values represent the means of three bulked samples
(one sample per treatment in each of three experiments).
Data were analyzed using orthogonal polynomial contrasts.
The effect of the hydrogen ion concentration was
significant and best described by a linear polynomial
(P < 0.05). The mean root length of L. sativa seedlings
at pH 4.0 and 25 C was significantly smaller than all
other inoculated treatments at 25 C (P < 0.05), but did
not differ from the noninoculated treatment at pH 4.0 and
25 C (P > 0.10). A second analysis was conducted to test
whether any treatments differed from non-treatment control
bioassays, which were made using fresh nutrient solution
samples (the mean of three replicates was 20.5 /urn) ; no
effects were observed (P > 0.10).

215
Table A-7. Effects of humidity and temperature on root
length of Lactuca sativa in bioassays from experiments on
disease caused by Fusarium solani f. sp. phaseoli in Vigna
radiata.
Humidity
(mm Hg, VPD)X
Temperature
(C)
Root length
(mm) y
Noninoculated
plants
Inoculated
plants
6.0
20
14.8Z
15.4
25
13.8
12.9
30
17.0
16.8
12.0
20
15.0
15.0
25
12.0
14.8
30
16.1
16.0
x Humidity was measured as the vapor pressure deficit (VPD),
in mm Hg; vapor pressure deficits of 6.0 and 12.0 mm Hg,
at 25 C, correspond to 75 and 45% relative humidities,
respectively.
y Root lengths of 40-45 four-day-old seedlings of L. sativa.
cv. 'Grand Rapids,' were measured using the Microcomp®
Video Image Analysis System.
z Samples from each replicate per treatment, for each
experiment, were bulked and processed as one sample.
Table values represent the means of two bulked samples
(one sample per treatment in each of two experiments).
Data were analyzed using orthogonal polynomial contrasts.
Effects of humidity, temperature, and inoculation were not
observed (P > 0.10) for root elongation of L. sativa,
except at 25 C and 12.0 mm Hg, where the root length of
the noninoculated treatment was significantly different
from most other treatments (P < 0.05). A second analysis
was conducted to test whether any treatments differed from
non-treatment control bioassays, which were made using
fresh nutrient solutions (the mean of two replicates was
15.4 /¿m) ; no effects were observed (P > 0.10).

216
Table A-8. Effects of light-quality and temperature on root
length of Lactuca sativa in bioassays from experiments on
disease caused by Fusarium solani f. sp. phaseoli on Vigna
radiata.
Lamp-type
Temperature
Root length
(mm) y
(C)
Noninoculated
plants
Inoculated
plants
Fluorescent
20
20.4Z
19.1
25
21.3
20.6
Metal halide
20
20.2
20.2
25
21.0
20.7
High pressure
20
20.4
20.5
sodium
25
20.6
19.8
y Root lengths
of 40-45, four-
-day-old seedlings
of L.
sativa. cv. 'Grand Rapids,' were measured using the
Microcomp® Video Image Analysis System.
z Samples from each replicate per treatment, for each
experiment, were bulked and processed as one sample.
Table values represent the means of three bulked samples
(one sample per treatment in each of three experiments).
Data were analyzed using an Analysis of Variance and least
squares mean-separation tests. Effects of temperature,
light quality, and inoculation were not observed
(P > 0.10) for root elongation of L. sativa. A second
analysis was conducted to test whether any treatments
differed from non-treatment control bioassays, which were
made using fresh nutrient solutions (mean of three
replicates was 20.3 /xm) ; no effects were observed
(P > 0.10).

APPENDIX B
DRY WEIGHTS AND SHOOT-ROOT RATIOS FROM
SPORE ATTACHMENT EXPERIMENTS

218
Table B-l. Effects of the hydrogen ion concentration at the time of plant inoculation on
disease caused by Fusarium solani f. sp. phaseoli on disease development in Vigna radiata.
PH
Plant dry weight (g)
Shoot-root
ratios^
Noninoculated
Inoculated
Noninoculated
Inoculated
4.0
0.76 az
0.38* a
4.5 a
5.3* a
5.0
0.74 a
0.61* b
4.8 a
6.0* a
6.0
0.72 a
0.59* b
4.8 a
5.9* a
7.0
0.67 a
0.69 b
5.1 a
4.8 a
y Shoot-
2 Table
-root ratios were estimated
values represent the means
using dry weights
of nine replicates
of plant tissues,
per treatment (three
replicates per
treatment in each of three experiments). Data were subjected to an orthogonal
polynomial contrast analysis. Effects of inoculation and a hydrogen ion-by-inoculation
interaction were observed and best fit by linear polynomial equations (P < 0.002).
Least-squares mean separation tests were used to examine pairwise comparisons between
appropriate treatments. Significant differences in comparisons between noninoculated
and inoculated plants at each hydrogen ion concentration are indicated by an asterisk
(P < 0.05). Treatments in columns followed by the same letter were not different
(P > 0.05).

Table B-2. Effects of inoculum density and temperature on
disease caused by Fusarium solani f. sp. phaseoli on Vigna
radiata.
Temperature
(C)
Ratios
of shoot
dry weight to root dry weight
Inoculum
density
(propagules/ml)
0
100
500
1000
2000
3000
20
3.0a
z 5.1b
6.5cd
7.5de
8.8e
8.2de
25
3.0a
4.0b
4.6b
4.5b
5.1b
5.2b
30
4.5a
5.3a
4.9a
4.8a
5.2a
5.3a
z Values represent the means of 28 replicates per treatment
(14 replicates per treatment in each of two experiments).
Shoot-root ratios were transformed to logs and then
subjected to an orthogonal polynomial contrast analysis;
table values are presented as detransformed numbers. The
effect of inoculum density was highly significant and best
described by a quadratic polynomial equation (P < 0.01).
Treatments within each temperature followed by the same
letter were not different (P > 0.05); differences were
based on least-squares mean separation tests. A
temperature by inoculum density interaction was observed
and was best fit by a linear-by-quadratic polynomial
equation (P < 0.01).

PLANT DRY WEIGHT (g)
220
INOCULUM DENSITY (PROPAGULES/ml)
Figure B-l. Effects of inoculum density of Fusarium solani
f. sp. phaseoli and temperature on the severity disease in
Viqna radiata. Bars indicate standard errors of the means.

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294.

BIOGRAPHICAL SKETCH
Andrew Conrad Schuerger was born on July 1, 1956, in
Cleveland, Ohio. Andrew attended the University of Arizona
in Tucson, AZ, between 1974 and 1979, where he earned a
Bachelor of Science degree with a double major in plant
pathology and entomology. Andrew completed his Master of
Science degree in plant pathology in 1981, also from the
University of Arizona. In 1982, he assumed the position of
senior plant pathologist at The Land, Epcot Center.
Begining in 1984, Andrew initiated studies at the University
of Florida for a doctoral degree in plant pathology.
243

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
f.
David J.v Mitchell, Chairman
Professor of Plant Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
R. Charudattan
Professor of Plant Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Barnes 0. Strandberg 0
Professor of Plant Pathology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
C /ff.
David M. Sylvi^/
Associate Professor of Soil
Science
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy. /'J
SkatÁ
August 1991 Dean, College of Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262
08285 436 4



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