Table Of ContentAN ABSTRACT OF THE THESIS OF
Pivarak Tanprasert for the degree of Master of Science in Horticulture
presented on May 13, 1996.
Title: Detection. Identification, and Antibiotic Treatment of Bacterial
Contaminants from Micropropagated Strawberries
Abstract approved: - - ,- -.
Barbara M. Reed
Bacterial contamination is one of the most important problems
both in plant tissue culture research and commercial laboratories. Early
detection of contaminants saves time, effort, and money. Strawberry
runner explants were screened for contaminants during June to August,
1994 and 1995. Most contaminants found were bacterial rather than
fungal. Bacterial contaminants from 22 contaminated genotypes were
isolated, purified, and identified to genus using standard biochemical
tests such as Gram's stain, King's B medium, starch hydrolysis, and
carbon source utilization (Biolog). From those 22 genotypes, 16 different
bacterial strains were isolated. The majority were Gram-negative, motile,
rod-shaped fluorescent pseudomonads. Xanthomonads,
enterobacterium, and other unidentified Gram-negative and Gram-
positive bacteria were also found. Minimal bactericidal concentrations
(MBCs) of single (Timentin, streptomycin sulfate, dihydrostreptomycin,
and gentamicin) and combinations of two and three (Timentin,
streptomycin sulfate, and gentamicin) were tested for inhibition of the
bacterial isolates. Combinations of Timentin, streptomycin sulfate, and
gentamicin killed all bacterial isolates tested. Fragaria mrginiana subsp.
glauca (S. Watson) Staudt and F. x ananassa Duch. Cv. Jucunda were
inoculated with Pseudomonas corrugata or Xanthomonas campestris pv.
vesicatoria then treated with a combination of Timentin, streptomycin
sulfate, and gentamicin. F. uirgirdana subsp. glauca died after
inoculation with either bacteria. Antibiotic treatments were 100%
effective in eliminating P. corrugata from 'Jucunda'/but only 23% of the
plants inoculated with X. campestris pv. vesicatoria were freed of the
bacteria.
Detection, Identification, and Antibiotic Treatments of Bacterial
Contaminants from Micropropagated Strawberries
by
Piyarak Tanprasert
A THESIS
submitted to
Oregon State University
In partial fulfillment of
the requirements for the
degree of
Master of Science
Completed May 13, 1996
Commencement June 1996
Master of Science thesis of Pivarak Tanprasert presented on May 16,
1996
APPROVED:
Major Professor, representing Horticulture
Chair of Department of Horticulture
Dean of Gradiu^attee SScchhio ol
I understand that my thesis will become part of the permanent collection
of Oregon State University libraries. My signature below authorizes
release of my thesis to any reader upon request.
Piyarak Tanprasert, Author
Acknowledgments
First of all, I would like to express my sincere gratitude to my
major advisor, Dr. Barbara M. Reed, for her kindness, encouragement,
understanding, etc. I know that I can never tell or express how much I
appreciate everything she has done for me. I can only say whit all my
heart that she is the most wonderful person. Some people who never
met Dr. Reed may think that I exaggerate, but I am more than sure that
anyone whoever met or talked with her even just once will understand
what I meant.
Not only do I have the best advisor, I also have the best committee
members, Dr. Larry W. Moore and Dr. Leslie Fuchigami. They both are
very kind, understanding, and open-minded. I do really appreciate them
for contributing their precious time to be my committee members.
I also would like to express my special thanks to Dr. Mike D.
Burgett for sparing his valuable time to serve as my graduate
representative. Although I had never met him before I asked him to be
my graduate representative, I have heard many good things about him
from all my friends for a long time.
My great appreciations are due to Dr. Patricia M. Buckley who has
taught me many things both in research and real life. I always miss
working with her. She is very nice, generous, and lovable.
Great appreciation is also due to Dr. Steve Giovannoni for allowing
me to use the Biolog database; Dr. Xintian for teaching me how to do the
Biolog test; Ms. Karen Keller for loaning me the multichannel
micropipette; and for Doug Gordon for helping me with Biolog reading.
Thanks to Kim Hummer, Carolyn Paynter, Joseph Postman, and
all the crew at the NCGR for their help and kindness. I am going to miss
all of them when I leave here.
Great acknowledgements are due to Dr. Siripom Nittayanggul who
served as my advisor in Thailand for her kindness and advice.
Special thanks are due to Peera Jitpraphai for his loving, caring,
and sharing all through years and years.
Special thanks are also due to Weerachai Siripunwarapom and
Chartchai Krittanai for helping and making suggestions to me all
through my graduate years, and also for invariably being my best
friends.
Thanks to Weerathep Pongprasert for helping me with statistical
analysis and being a good neighbor. Thanks also to P'Pop for being a
very good neighbor.
Many thanks are due to Ped, Pa, Huang, Lin, Tai, Golf, Fon, Pop,
Yong, T-yai, P'Jeab, P'Moo, Lek, Vit, and all other Thai friends both here
in America now and in Thailand for being my very good friends.
Thanks to my former roommates, PTBee, Yu, and Phon; and to my
present roommates Sa and Mam for encouragement and cooking for me.
Thanks to Xiaoling Yu, Dennis Yeo, Jie Luo, Yongjian Chang, and
all other friends in OSU for being my friends.
Last but never least, my highly love, respect, and great
appreciations are due to my mom, dad, brothers, and sisters.
I am glad that I have a chance to express some of my love and
appreciation to everyone, but I regret that I cannot state all the names of
my beloved friends or be able to express all my appreciations through
these limited pages of acknowledgements. I wish you all will understand
how much I love and appreciate all of you to be my friends.
I feel like I am the luckiest person to meet and know so many
wonderful people. I don't know what I was bom for, but I do know that I
am very glad to be bom and to be who I am.
"WHOEVER SAYS LOVE CANNOT DO MUCH,
I SAY LOVE CAN DO ANYTHING!"
CONTRIBUTION OF AUTHOR
Dr. Barbara M. Reed participated the entire experimental designs,
analyses, discussions, support, and critical editing.
TABLE OF CONTENTS
Page
Chapter 1. Introduction and Literature Review 1
Micropropagation of Strawberry 1
Culture media 1
Plant growth regulators 4
Rooting 4
Acclimatization 5
Bacterial Contamination in Micropropagated Plants 6
Sources of bacterial contamination 6
Explants 7
Operators 8
Mites and thrips 9
Inefficient sterilization techniques 9
Prevention of bacterial contaminants 10
Detection 11
Identification and characterization 12
Antibiotic treatments 13
References 17
TABLE OF CONTENTS (Continued)
Page
Chapter 2. Detection and Identification of Bacterial Contaminants 23
in Strawberry Runner Explants
Abstract 24
Introduction 25
Material and methods 28
Plants material used 28
Plants response to submerged growth 28
Detection of contaminants 29
Isolation and purification of bacteria 29
Diagnostic tests for bacterial characteristics 29
Standard bacterial cultures 30
Carbon substrate utilization 30
Results and discussion 31
Plant response to submerged growth 31
Detection of contaminants 31
Characterization and identification of bacteria 33
Standard bacterial cultures 40
Standard biochemical tests 41
Carbon substrate utilization (Biolog) test 41
Conclusions 43
References 45
Description:Most contaminants found were bacterial rather than fungal. Bacterial contaminants from 22 contaminated genotypes were isolated, purified, and identified to genus using standard biochemical tests such as Gram's stain, King's B medium, starch hydrolysis, and carbon source utilization (Biolog).
