Effects of temperature and hydrogen ion concentration on disease caused by Fusarium solani f. sp. phaseoli in Vigna radi...

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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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xiii, 243 leaves : ill. ; 29 cm.
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Schuerger, Andrew Conrad, 1956-
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Thesis (Ph. D.)--University of Florida, 1991.
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Includes bibliographical references (leaves 221-242).
Statement of Responsibility:
by Andrew Conrad Schuerger.
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Typescript.
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Vita.

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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