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Genetic variation and virulence
differentiation of Pantoea stewartii
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GENETIC VARIATION AND VIRULENCE DIFFERENTIATION OF
PANTOEA STEWARTII ISOLATES FROM THE EASTERN UNITED STATES
By

Matthew Ryan Durkin

A THESIS
Submitted to
Michigan State University

in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Plant Pathology

2002

ABSTRACT

GENETIC VARIATION AND VIRULENCE DIFFERENTIATION OF
PANTOEA S TE WARTII ISOLATES FROM THE EASTERN UNITED STATES

By
Matthew Ryan Durkin

Stewart’s bacterial wilt is a sporadic disease of sweet corn caused by the bacterium
Pantoea stewam'i and can be responsible for crop losses of up to 100%. Outbreaks of
Stewart’s bacterial wilt are currently thought to be dependent on the ability of the corn ﬂea
beetle (Chaetocnema pulicaria) to vector P. stewartii. In this research, genomic ﬁngerprints
were generated using the repetitive sequence-based polymerase chain reaction (rep-PCR)
with the BOX and REP primers to differentiate between 84 isolates of P. stewartii that
ranged in collection date (1967-2001) and location (Michigan, Illinois, Iowa, Ohio, North
Carolina, Kentucky, Indiana). The resulting genomic ﬁngerprints for each isolate were
separated into four different strain types. Strain types had a similarity of 94-97% suggesting
limited genetic diversity among isolates. These genetic data may be useful when studying
future epidemics.

Virulence evaluations of P. stewartii isolates representative of each strain were also
performed on four sweet corn varieties ranging in susceptibility to Stewart’s bacterial wilt
from susceptible to resistant. There was no relation between a particular rep-PCR strain type
and virulence, but individual isolates were more virulent on certain sweet corn varieties.
Pathogen virulence assessment may help with forecasting and prevention of future outbreaks.

Three collection methods for P. stewartii isolates (baiting with a susceptible sweet
corn variety, sweeping for beetles, and scouting grower’s ﬁelds) were also compared. During
a non-epidemic year, baiting with a susceptible variety was the only method where P.

stewartii isolates were successfully gathered.

To my parents, family, and my soon to be wife, Emily .....
Without you, this wouldn 't be possible ......

iii

ACKNOWLEDGMENTS

I would like to acknowledge my committee members, Dr. Willie Kirk, Dr. James
Smith, Dr. Dennis Fulbright, and Dr. Andrew Jarosz. Willie, I am very grateful you gave me
the opportunity to explore the world of plant pathology. Thank you for allowing the perfect
balance of independence and structure during my research. Also, thank you for being my
friend, I enjoyed the time we spent out of the laboratory, especially on the golf course. You
proved to me that although the Scottish invented the game, that does not mean that they all
can play it. Jim, thanks for the long conversations we had about what my research really
meant. I really enjoyed the two courses that I was able to take with you as the professor. Dr.
Fulbright, thank you for discussing my ‘quick questions’ with me. Although they may have
been quick, your responses always required me to step back and reanalyze most situations.
Dr. Jarosz, thank you for demanding the best. I enjoyed our conversations and your views
strengthened my research.

Thanks to everyone in Dr. Willie Kirk’s laboratory, Rob Schafer, Jeff Stein, Sara
Shaw, Dr. Kathleen Baker, Devan Berry, and Brandon Dunlap for making my research
enjoyable both in and out of the laboratory. Thank you, Carmen Medina-Mora for teaching
me the an of PCR. Special thanks to Luis Velasquez for his microbiology mentoring and
friendship.

And last but not least, I would like to thank my ﬁancee, my strength, my friend, my
love, Emily Tompkins, for proving encouragement and support when it did not seem worth
the trouble anymore. When I didn’t believe in myselfI always knew she believed in me and
my abilities. I love you Emily.

Thank you to everyone. Without your help and support I would not have reached my

goal

iv

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ vii
LIST OF FIGURES .......................................................................................................... viii
CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
Introduction ............................................................................................................. 1
Deﬁnitions ............................................................................................................... 2
Stewart’s Bacterial Wilt .......................................................................................... 3
Taxonomy ................................................................................................................ 4
The Vector: Chaetocnema pulicaria ....................................................................... 6
Stewart’s Bacterial Wilt Epidemics ........................................................................ 8
Bacterial Population Analysis ............................................................................... 10
Objectives of this Research ................................................................................... 12
CHAPTER 2
MATERIALS AND METHODS
Isolates Studied ..................................................................................................... 14
Michigan Isolate Collection Techniques ............................................................... l4
Isolation Techniques ............................................................................................. 21
rep-PCR differentiation between members of the genus Erwinia and Pantoea 22
Identification of Isolates (Gram Stain, Pathogenicity, BOX-PCR) ...................... 23
rep-PCR using REP-PCR and BOX-PCR ............................................................. 25
Virulence Assessment Protocol (Leaf Symptom Analysis) .................................. 26
Virulence Assessment Protocol (Colony Forming Units in
Second Leaf Analysis) .......................................................................................... 29
Data Analysis ........................................................................................................ 30
CHAPTER 3
RESULTS
Michigan Isolate Collection .................................................................................. 32
rep-PCR (BOX-PCR) differentiation between
members of the genus Erwinia and genus Pantoea .............................................. 32
BOX-PCR genetic variation of P. stewartii strains ............................................... 35
REP-PCR genetic variation of P.stewartii strains ................................................. 35
Determination of strains based on BOX-PCR and REP-PCR .............................. 38
Virulence Assessment (Leaf Symptom Analysis) ................................................. 38
Virulence Assessment (Colony Forming Units in
the Second Leaf Analysis) ..................................................................................... 47
CHAPTER 4
DISCUSSION ................................................................................................................... 54
REFERENCES .................................................................................................................. 62

APPENDIX A

OPTIMIZATION OF BOX PRIMER FOR REP-PCR (EXAMPLE) .............................. 67
APPENDIX B

OPTIMIZATION OF REP PRIMER FOR REP-PCR (EXAMPLE) ............................... 69
APPENDIX C

IDENTIFICATION OF CFU USING REP-PCR AND THE BOX PRIMER .................. 71
APPENDIX D

REP-PCR USING THE BOX PRIMER GELS WITH

84 ISOLATES OF P.STEWARTII ..................................................................................... 73
APPENDD( E

REP-PCR USING THE BOX PRIMER GELS WITH

84180LATES OF P. STE WARTI ..................................................................................... 90

vi

LIST OF TABLES

Table 1. Bacterial isolates used in this research for rep-PCR analysis and virulence
differentiation on sweet corn ............................................................................................. 15

Table 2. Varieties of sweet corn used in virulence assessment protocol .......................... 27

Table 3. Pantoea stewartii strain types used as inoculum in virulence assessment
protocol .............................................................................................................................. 28

Table 4. Pantoea stewartii isolates collected from Michigan during the 2001 growing
season ............................................................................................................................... 33

Table 5. Area under the disease progress curve (AUDPC) for Stewart’s bacterial wilt
symptoms on individual leaves (AUDPCU, AUDPCLz, AUDPCu), on leaves 2 and 3
(AUDPCm), and leaves 1, 2, and 3 (AUDPCLH) for four varieties (combined data)

of sweet corn which were rated for 4 days starting 3 days after inoculation at the 3-1eaf
stage... ............................................................................................................................... 45

Table 6. Area under the disease progress curve (AUDPC) for Stewart’s bacterial wilt

symptoms on the combination of leaves 2 and 3 (AUDPCm) on four separate varieties of
sweet corn which were rated for 4 days starting 3 days after inoculation ........................ 46

vii

LIST OF FIGURES

Figure 1. Southern portion of Michigan’s Lower Peninsula showing the twelve locations where
isolates of Pantoea stewartii were sought. The roman numerals represent the regions and the
lettered stars represent the closest city to the ﬁelds. I-A-Fremont; I-B-Grand Rapids; I-C-Byron
City; II-A-Romeo; II-B-Almont; II-C-Riley Center; III-A-Chelsea (ﬁeld 1) III-B-Chelsea (ﬁeld
2); III-C-Monroe; IV-A-Dowagiac; IV-B-Decatur; IV-C-Marcellus ............................................ 20

Figure 2. Sweet corn plant at the 2-3-leaf grth stage. The needle was inserted at the arrow
marked by the coleoptile label ....................................................................................................... 24

Figure 3. BOX-PCR generated genomic ﬁngerprint, on a 1.5% agarose gel, of Pantoea and
Erwinia species. The numbered brackets below indicate the isolate of Pantoea or Erwinia that is
being analyzed. Each strain is repeated in adjacent lanes and represented by the same number.
The two lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. 2-Erwinia chrysanthemi; 3-
Erwinia carotovora; 4-Erwinia amylovora (Es 110); 5-E. amylovora (Es 321); 6-5. amylovora
(Es MR1); 7-E. amylovora (PSLN); 8-E. amylovora (CFB Pr). Lanes, 9-Pantoea stewartii (Il-Ps
1) and 10-Pantoea stewartii (ATCC-29227) are differentiated by a 650 bp band that is absent in
lane 9, but present in lane 10 ......................................................................................................... 34

Figure 4. BOX-PCR generated genomic ﬁngerprint, on a 1.5% agarose gel, of 15 Pantoea
stewartii strains that were used in the virulence trial. The numbered lines with arrows below
indicate the isolate of P. stewartii that is being analyzed. The three lanes labeled with an ‘L’
contain a 1 Kb DNA Ladder. Strains missing the 650-bp band - Lane 2-Illinois (Ps-VI-A-CP-l-
1); 3-Illinois (Ps-V-A-CP-l-l); 13-Iowa (Es 9245); 15- Michigan (Ps-II-B-CP-l); 16- Illinois (Il-
Ps 1). Strains with the 650-bp band- Lane 4-Iowa (SW 87-2); 5-Illinois (SW 45); 6-Ohio (SW
1); 7-Iowa (SW 87-19); 8-Iowa (SW 87-6); lO-Iowa (Es 9208); ll-Iowa (Es 9211); 12-Iowa ( Es
9230); 14-Illinois (SS 104); l7-ATCC-29227 ............................................................................... 36

Figure 5. REP-PCR generated genomic ﬁngerprint, on a 1.5% agarose gel, of 15 Pantoea
stewartii strains that were used in the virulence trial. The numbered lines with arrows below
indicate the isolate of P. stewartii that is being analyzed. The three lanes labeled with an ‘L’
contain a 1 Kb DNA Ladder. Strains missing the 380-bp band - Lane 6-Ohio (SW1); 7-Iowa (SW
87-19); 8-Iowa (SW 87-6); 12-Iowa (Es 9230); 13-Iowa (Es 9245); l4-Illinois (SS 104). Strains
with the 380-bp band- 2-Illinois (Ps-VI—A-CP-l-l); 3-Illinois (Ps-V-A-CP-l-l); 4-Iowa (SW 87-
2); 5-Illinois (SW 45); 10-Iowa (Es 9208); ll-Iowa (Es 9211); 15- Michigan (Ps-II-B-CP-l); 16-
Illinois (Il-Ps 1); 17-ATCC-29227 ................................................................................................ 37

Figure 6. The rep-PCR strain type (1, 2, 3, 4) distribution of 84 Pantoea stewartii isolates from
the eastern United States (Table 1) ................................................................................................ 39

Figure 7 (A-C). Progression of Stewart’s bacterial wilt symptoms (scale 0-3 as described in text;
brieﬂy 0: no symptoms, 3: complete necrosis of leaf) on individual leaves (Figure A-C, leaf
position 1-3) from four varieties of sweet corn [Jubilee, Native Gem, Buckeye, Ambrosia
(individual points represent the average severity score of varieties combined)] after inoculation
with selected isolates of P. stewartii from Eastern USA (listed in Table 2).

+ Negative control (No bacteria) ;-v-‘ IlEsl;—v-- II-B-CP-l .-—l- - SSlO4 .
—D— E39245 ;—9- Es 9230 ;-<>— Es 9211 .”“"“ Es 9208 .-A-- SW 87-6.

viii

—..- SW87-19.-—0--SW1.—O- SW45 .—0— sws7-2.
—'— V-A-CP-l-l -'“""V VI'A'CP'I'I ................................................................................ 40

Figure 8. Estimate of the area under the disease progress curve on leaves 1, 2, and 3 (AUDPCU,
AUDPCLz, AUDPCU) of the combined disease symptoms caused by 15 separate Pantoea
stewartii strains on four different varieties of sweet corn ................................................................. 43

Figure 9. Estimate of the area under the disease progress curve of leaves 2 and 3 (AUDPCm) for
the combined disease symptoms caused by 15 separate Pantoea stewartii strains on four

individual varieties of sweet corn which were needle inoculated near the coleoptile and allowed to
incubate for six days. Bars followed by the same letter were not signiﬁcantly different at the

p=0.05 level of probability ................................................................................................................ 44

Figure 10. Average CFU (108) from a 115 mm2 section of leaf 2 (samples were serially diluted to
10’s) for each of the four varieties of sweet corn six days after inoculation with 15 different

isolates of P. stewartii. When inoculated with isolates of P. stewartii, plants were in a random
complete block design with eight replications. Bars followed by the same letter were not
signiﬁcantly different at the p=0.05 level of probability ................................................................... 48

Figure 11. Average CFU (108)/ 115 mm2 of leaf tissue (samples were serially diluted to 10's) in
leaf two of Jubilee sweet corn plants six days after inoculation with 15 different isolates of P.
stewartii. Brackets below (14) indicate the reme strain type of each isolate. The same
bracketed letters above bars were not signiﬁcantly different at p<0.05 level of probability ............. 50

Figure 12. Average CFU (108)/ 115 mm2 of leaf tissue (samples were serially diluted to 10's) in
leaf two of Native Gem sweet corn plants six days after inoculation with 15 different isolates of

P. stewartii. Brackets below (14) indicate the rep-PCR strain type of each isolate. The same
bracketed letters above bars were not signiﬁcantly different at p<0.05 level of probability ............. 51

Figure 13. Average CFU (108) I 115 mm2 of leaf tissue (samples were serially diluted to 10") in
leaf two of Buckeye sweet corn plants six days after inoculation with 15 different isolates of P.
stewartii. Brackets below (14) indicate the reme strain type of each isolate. The same
bracketed letters above bars were not signiﬁcantly different at p<0.05 level of probability ............. 52

Figure 14. Average CFU (10") I 115 mm2 of leaf tissue (samples were serially diluted to 10") in
leaf two of Ambrosia sweet corn plants six days after inoculation with 15 different isolates of P.
stewartii. Brackets below (1-4) indicate the rep-PCR strain type of each isolate. The same
bracketed letters above bars were not signiﬁcantly different at p<0.05 level of probability ............. 53

Figure 15. Optimization of Rep-PCR using the BOX primer generated genomic ﬁngerprint

of Pantoea stewartii strains (ATCC-29227 and Il-Ps 1). The numbered brackets below indicate

the strain of P. stewanii and levels of H20 that is being analyzed. Each strain and H20 alteration

is repeated in adjacent lanes and represented by the same number. The three lanes labeled with

an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2-Il-Ps 1 (12.25 pl of H20); 3-ATCC-29227 (12.25 pl

of H20); 4-Il-Ps l (12.30 pl of H20); 5 ATCC-29227 (12.30 p1 of H20); 6-Il-Ps l (12.35 pl of

H20); 7-ATCC-29227 (12.35 pl of H20); 8-Il-Ps l (12.40 pl of H20); 9-ATCC-29227 (12.40 pl of
H20); 10-Il-Ps 1 (12.45 pl of H20); 11-ATCC-29227(12.45 pl of H20); 12-Il-Ps 1 (12.50 pl of
H20); 13-ATCC-29227 (12.50 pl of H20); 14-Il-Ps 1 (12.55 pl of H20); 15-ATCC-29227 (12.55

pl of H20) .......................................................................................................................................... 68

ix

Figure 16. Optimization of Rep-PCR using the REP primer generated genomic ﬁngerprint of
Pantoea stewartii strains (ATCC-29227 and Il-Ps l). The numbered brackets below indicate the
strain of P. stewartii and levels of H20 that are being analyzed. Each strain and H20 alteration is
repeated in adjacent lanes and represented by the same number. The three lanes labeled with an

‘L’ contain a 1 Kb DNA Ladder. Lane 2-ATCC-29227 (12.45 pl of H20); 3-II-Ps 1 (12.45 p1 of
H20); 4 ATCC-29227 (12.50 pl of H20); 5-Il-Ps l (12.50 pl of H20); 6-ATCC-29227 (12.55 pl of
H20); 7-Il-Ps l (12.55 pl of H20); 8-ATCC-29227 (12.60 pl of H20); 9-Il-Ps 1 (12.60 pl of H20);
lO—ATCC-29227 (12.65 pl of H20); 11-Il-Ps 1 (12.65 pl of H20) .................................................... 70

Figure 17. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of 15 Pantoea stewartii strains that were randomly selected as checks to conﬁrm that the
colonies counted in the virulence trial were P. stewartii. The numbered lines with arrows below
indicate the isolate of P. stewartii that is being analyzed. The three lanes labeled with an ‘L’
contain a 1 Kb DNA Ladder. Strains missing the 650-bp band - Lane 2-Illinois (Ps-VI-A-CP—l-

l); 3-Illinois (Ps-V-A-CP-l-l); 13-Iowa (Es 9245); 15- Michigan (II-B-CP-l); 16- Illinois (Il-Ps

1). Strains with the 650-bp band- Lane 4-Iowa (SW 87-2); 5-Illinois (SW 45); 6-Ohio (SW 1);
7-Iowa (SW 87-19); 8-Iowa (SW 87-6); 10-Iowa (Es 9208); ll-Iowa (Es 9211); 12-Iowa( Es
9230); 14-Illinois (SS 104); l7-ATCC-29227 ................................................................................... 72

Figure 18. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Illinois (V III-A-CP-3-
l); 4-Illinois (VI-A-CP-2-l); 5-Illinois (V-A-CP-4-2); 6-Illinois (V III-A-CP-3-2); 7-Michigan (II-
B-CP-l); lO-Illinois (V-A-CP-l-2); ll-Illinois (VI-A-CP-2-3); 12-Illinois (VI-A CP-1-3); 13-
Illinois (V I-A-CP-1-2); 14-Illinois (V I-A-CP-l-l) ....................................................................... 74

Figure 19. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- Illinois-control (Il-Ps-l) and ATCC-29227. Lane 8 and 9 also
contain strain Illinois-control (Il- Ps-l) and ATCC-29227, respectively. 3-Iowa (SW 87-1); 4-
Iowa (SW 87-2); S-Ohio (SW1); 6-Illinois (V -A—CP-1-1); 7-Illinois (V-A-CP-3-2); 10-Illinois

(V -A-CP-3-1); 11-Illinois (V -A-CP-4-1); 12-Iowa (SW 87-3); 13-Iowa (SW 87-4); 14-Illinois
(VI-A-CP-4-1) ............................................................................................................................... 75

Figure 20. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewanii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control(Il-Ps-1), respectively. 3-Iowa (SW 87-23); 4-
Iowa (SW-87-22); 5-Iowa (SW-87-21); 6-Iowa (SW-87-20); 7-Iowa (SW-87-l9); lO-Iowa (SW-
87-18); ll-Iowa (SW-87-9); 12-Iowa (SW-87-7); 13-Iowa (SW-87-25); 14-Iowa (87-24) ......... 76

Figure 21. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same

number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control(Il-Ps-1), respectively. 3-Iowa (SW-87-17); 4-
Iowa (SW-87-16); 5-Iowa (SW-87-15); 6-Iowa (SW-87-l 3); 7-Iowa (SW 87-12); lO-Iowa (SW
87-11); ll-Iowa (SW 87-10); 12-Iowa (SW-87-8); 13-Iowa (SW-87-5); l4-Iowa (SW-87-6) 77

Figure 22. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es 9225); 4-Iowa
(Es 9230); 5-Iowa (Es 9243); 6-Iowa (Es 9244); 7-Iowa (Es 9245); lO-Iowa (Es 9222); 11-Iowa
(Es 9221); l2-Iowa (Es 9203); 13-Iowa (Es 9204); 14-Iowa (Es 9205) ........................................ 78

Figure 23. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- Illinois-control (Il- Ps-l) and ATCC-29227. Lane 8 and 9 also
contain strain Illinois-control (Il- Ps-l) and ATCC-29227, respectively. 3-Illinois (SS 104) . 4-
North Carolina (SW 39); 5-Iowa (Es 9250); 6-Iowa (Es 9256); 7-Tennessee (SW 36); lO-Iowa
(Es 9253); ll-Iowa (Es 9245); 12-Indiana (SW 14); 13-Iowa (Es 9247); 14-Illinois (SW 45) 79

Figure 24. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5 % agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls - Illinois-control (Il-Ps-l) and ATCC-29227. Lane 8 and 9 also
contain strain Illinois-control (Il-Ps—l) and ATCC-29227, respectively. 3-Indiana (SW 13); 4-
Ohio (SW 2); 5-Ohio (Rif EsA); 6-Iowa (Es 9257); 7- Ohio (SW 1); lO-Iowa (Es 9254);
11-Kentucky (SW 19); 12-Iowa (Es 9248); 13-Iowa (Es 9249); l4-Iowa (9246) ......................... 80

Figure 25. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es 9203); 4-Iowa
(Es 9204); 5 Iowa (Es 9205); 6-Iowa (Es 9206); 7-Iowa (Es 9207); 10-Iowa (Es 9208); 1 1-Iowa

(Es 9209); 12-Iowa (Es 9211); 13-Iowa (Es 9210); 14-Iowa (Es 9201) ............................................ 81

Figure 26. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es 9223); 4-Iowa
(Es 9222); 5-Iowa (Es 9221); 6-Iowa (Es 9213); 7-Iowa (Es 9212); 10-Iowa (Es 9264); ll-Iowa

(Es 9244); 12-Iowa (Es 9243); l3-Iowa (Es 9246); 14-Iowa (Es 9230) ............................................ 82

xi

Figure 27. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- Illinois-control (Il-Ps-l) and ATCC-29227. Lane 8 and 9 also

contain strain Illinois-control (Il-Ps-l) and ATCC-29227, respectively. 3-Illinois (SW 45); 4-
Kentucky (SW 19); S-Ohio (GAL E8); 6-Ohio (DC 130); 7-Ohio (DC 160); lO-Ohio (MU 51);
ll-Indiana (SW 13); 12-Indiana (SW 14); 13-Ohio (DC 283); l4-Michigan (H—B-CP-7) ............... 83

Figure 28. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es 9249); 4-Iowa
(Es 9247); 5-Iowa (Es 9248); 6-Iowa (Es 9250); 7-Iowa (Es 9253); lO-Iowa (Es 9256); ll-Ohio
(Rif Es 9A); 12-Illinois (SS 104); 13-Iowa (Es 9264); l4-Iowa (Es 9257) ....................................... 84

Figure 29. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es 9213); 4-Iowa.
(Es 9212); 5-Iowa (Es 9211); 6-Iowa (Es 9210); 7-Iowa (Es 9206); 10-Iowa (Es 9207); ll-Iowa

(Es 9208); 12-Iowa (Es 9209); 13-Iowa (SW 87-26); 14-Iowa (Es 9201) ........................................ 85

Figure 30. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbers below the arrows indicate the strain of Pantoea
stewartii that was analyzed. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder.
Lanes 2 and 3 contain the positive controls- Illinois-control (Il-Ps-l) and ATCC-29227,
respectively. Lane 17 contains the ATCC-29227 control. Lane 28 and 29 also contain strain
Illinois-control (Il-Ps-l) and ATCC-29227, respectively. 4-Iowa (SW 87-11); 5-Iowa (SW 87-
12); 6-Iowa (SW 87-13); 7 Iowa (SW 87-15); 8 Iowa (SW 87-1); 9-Ohio (DC 130); 10-Ohio (DC
160); ll-North Carolina (SW 39); l2-Illinois (SW 45); 13-Tennessee (SW 36); 14-Kentucky (SW
19); 15-Iowa (SW 87-16); 18-Iowa (SW 87-17); 19-Iowa (SW 87-18); 20-Iowa (SW 87-19); 21-
Iowa (SW 87-20); 22-Iowa (SW 87-21); 23-Iowa (SW 87-26); 24-Iowa (SW 87-25); 25-Iowa
(SW 87-24); 26-Iowa (SW 87-23); 27-Iowa (SW 87-22) .................................................................. 86

Figure 31. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbers below the arrows indicate the strain of Pantoea
stewartii that was analyzed. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder.

Lane 2 contains the positive control- ATCC-29227. Lane 13 contains the Illinois control (11 Ps-l).
Lane 21 contains ATCC-29227. 3-Ohio (DC 283); 4-Ohio (SW 1); 5-Ohio (MU 51); 6-Ohio

(GAL E8); 7-Indiana (SW 14); 8-Indiana (SW 13); 9-Ohio (SW 2); 10-Iowa (SW 87-2); 1 1-Iowa
(SW 87-3); l4-Iowa (SW 87-4); 15-Iowa (SW 87-5); l6-Iowa (SW 87-6); 17-Iowa (SW 87-7);
18-Iowa (SW 87-8); l9-Iowa (SW 87-9); 20-Iowa (SW 87-10) ....................................................... 87

Figure 32. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose
gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same

xii

number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 10

contain the positive controls- Illinois-control (Il-Ps-l) and ATCC-29227. Lane 5 and 6 also

contain strain Illinois-control (Il-Ps-l) and ATCC-29227, respectively. 3-Illinois (V-A-CP-4-l);
4-Illinois (V III-A-CP-3-2); 7-Illinois (VIII-A-CP-3-l); 8-Illinois (V I-A-CP-2-1); 9-Illinois (V I-
A-CP-4-1) .......................................................................................................................................... 88

Figure 33. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Illinois (VI-A-CP-l-2);
4-Illinois (VI-A-CP-1-3); 5-Illinois (V-A-CP4-2); 6-Michigan (II-B-CP-S); 7-Illinois (VI-A-CP-
1-3); lO-Illinois (V-A-CP-3-2); ll-Illinois (V-A-CP-l-l); 12-Illinois (V-A-CP-3-1); l3-Illinois
(V-A-CP-l-Z); 14-Illinois (V I-A-CP-l-l) ......................................................................................... 89

Figure 34. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Illinois (VIII-A-CP-3-

l); 4-Illinois (VI-A-CP-2-1); 5-Illinois (V-A-CP-4-2); 6-Illinois (V III-A-CP-3-2); 7-Michigan (II-
B-CP-l); lO-Illinois (V-A-CP-l-2); ll-Illinois (VI-A-CP-2-3); 12-Illinois (V I-A CP-l-3); 13-
Illinois (V I-A-CP-1-2); l4-Illinois (V I-A-CP-l-l) ........................................................................... 91

Figure 35. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (SW 87-1); 4-

Iowa (SW-87-2); 5-Ohio (SW 1); 6-Illinois (V -A-CP-l-1); 7-Illinois (V-A-CP-3-2); lO-Illinois
(V-A-CP-3-l); ll-Illinois (V-A-CP-4-l); 12-Iowa (SW-87-3); 13-Iowa (SW-87-4); 14-Illinois

(V I-A-CP-4-l) ................................................................................................................................... 92

Figure 36. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (SW 87-23); 4-
Iowa (SW-87-22); 5-Iowa (SW-87-15); 6-Iowa (SW 87-13); 7-Iowa (SW-87-12); 10-Iowa (SW-
87-11); ll-Iowa (SW 87-10); 12-Iowa (SW-87-8); 13-Iowa (SW-87-5); 14-Iowa (SW 87-6) ......... 93

Figure 37. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (SW 87-17); 4-

xiii

Iowa (SW—87-l6); 5-Iowa (SW-87-21); 6-Iowa (SW 87-20); 7-Iowa (SW-87-l9); lO-Iowa (SW-
87-18); ll-Iowa (SW 87-9); 12-Iowa (SW-87-7); 13-Iowa (SW-87-24); 14-Iowa (SW 87-25) ....... 94

Figure 38. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9257); 4-Iowa
(Es 9256); 5-Iowa (Es 9253); 6-Iowa (Es 9250); 7-Iowa (Es 9249); 10-Iowa (Es 9248); ll-Iowa

(Es 9247); 12-Kentucky (SW 19); l3-Indiana (SW 2); l4-Indiana (SW 13) .................................... 95

Figure 39. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9213); 4-Iowa
(Es 9212); 5-Iowa (Es 9211); 6-Iowa (Es 9210); 7-Iowa (Es 9206); 10-Iowa (Es 9207); ll-Iowa

(Es 9208); 12-Iowa (Es 9209); l3-Iowa (SW 87-26); l4-Iowa (Es 9201) ........................................ 96

Figure 40. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9249); 4-Iowa
(Es 9247); 5-Iowa (Es 9248); 6-Iowa (Es 9250); 7-Iowa (Es 9253); 10-Iowa (Es 9256); ll-Ohio
(Rif Es 9A); lZ-Illinois (SS 104); l3-Iowa (Es 9264); 14-Iowa(Es 9257) ....................................... 97

Figure 41. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9243); 4-Iowa
(Es 9240); 5-Iowa (Es 9225); 6-Iowa (Es 9230); 7-Iowa (Es 9222); 10-Iowa (Es 9221); ll-Iowa

(Es 9213); 12-Iowa (Es 9244); l3-Iowa (Es 9245); l4-Iowa (Es 9246) ............................................ 98

Figure 42. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,
of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Ohio (DC 283); 4-Ohio
(DC 160); S-Ohio (DC 130); 6-Iowa (SW 87-8); 7-Iowa (SW-87-7); lO-Iowa (SW-87—6); 11-
Indiana (SW 14); 12-Tennessee (SW 36); l3-North Carolina (SW-39); 14-Illinois (SW 45) .......... 99

Figure 43. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose

gel, of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea
stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the

xiv

same number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and

15 contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also
contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Michigan (II-B-CP-l);
4-Iowa (SW-87-4); 5-Iowa (SW-87-5); 6-Iowa (SW 87-18); 7-Iowa (SW-87-l7); lO-Iowa
(SW-87-3); ll-Iowa (SW 87-2); lZ-Michigan (II-B-CP-7); l3-Iowa (SW-87-1); 14-Michigan (II-
B-CP-S) ........................................................................................................................................ 100

Figure 44. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9246); 4-Iowa
(Es 9212); 5-Iowa (Es 9210); 6-Iowa (Es 9208); 7-Iowa (Es 9207); lO—Iowa (Es 9206); l 1-Ohio

(SS 104); 12-Iowa (Es 9264); 13-Ohio (DC 283); l4-Ohio (MU 51) ............................................... 101

Figure 45. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Illinois(VI-A-CP-l-1);
4-(SW 45); 5- (SW 39); 6-Illinois (V-A-CP-3-2); 9-Illinois (V III-A-CP-3-2); lO-Illinois (V -A-
CP-4-2); ll-Illinois (V I-A CP-2-1); 12-Illinois (V I-A-CP—4-l) ....................................................... 102

Figure 46. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Illinois (VI-A-CP—2-3);
4-Illinois (V -A-CP-1-2); 5- Illinois (V -A-CP-3-1); 6-Illinois (V -A-CP-1-l); 9-Illinois (V -A-CP-
4-1); lO-Illinois (V I-A-CP-1-3); ll-Illinois (VI-A CP-1-2); 12-Illinois (V III-A-CP-3-1) ............... 103

Figure 47. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Ohio (GAL E8); 4-Ohio
(MU 51); 5-Illinois (SW 45); 6-North Carolina (SW 39); 7-Tennessee (SW-36); 10-Indiana (SW

14); ll-Ohio (DC 130); 12-Ohio (DC 160); l3-Ohio (DC 283); 14-Ohio (SW 1) ........................... 104

Figure 48. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewam'i strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Ohio (GAL E8); 4-Ohio
(MU 51); 5-Illinois (SW 45); 6- North Carolina (SW 39); 7-Tennessee (SW-36); lO-Indiana (SW

14); ll-Ohio (DC 130); l2-Ohio (DC 160); 13-Ohio (DC 283); l4-Ohio (SW 1) ........................... 105

XV

Figure 49. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (87-13); 4-Iowa
(87-15); 5-Iowa (87-16); 6-Iowa (87-20); 9-Iowa (87-19); 10-Iowa (87-22); 11-Iowa (87-21); 12-
Iowa (87-6) ........................................................................................................................................ 106

Figure 50. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5% agarose gel,

of Pantoea stewartii strains. The numbered brackets below indicate the strain of Pantoea

stewartii that was analyzed. Each strain is repeated in adjacent lanes and represented by the same
number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15

contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also

contain strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (87-7); 4-Iowa
(87-8); 5-Iowa (87-18); 6-Iowa (87-17); 9-Iowa (87-9); 10-Iowa (87-10); ll-Iowa (87-11); 12-

Iowa (87-12) ...................................................................................................................................... 107

xvi

CHAPTER 1

INTRODUCTION AND LITERATURE REVIEW

Bacterial plant pathogens are a diverse group of prokaryotic organisms able to
infect and kill plants by causing diseases including blights, scabs, wilts, leaf spots,
cankers, and soft rots. Each phytopathogenic bacterial species has unique characteristics
that can be used to separate it from other organisms. Phytopathogenic bacteria can be
either Gram positive or Gram negative and may be aerobic, anaerobic, or facultatively
anaerobic (Gerhardt, 1981). The cell shape of phytopathogenic bacteria are generally
bacilli, but there are other morphologies, such as cocci, coryneform, and ﬁlaments
(Gerhardt, 1981). When allowed to grow as single colonies on solid medium, such as
nutrient broth agar, colony morphology can be round, wavy edged, concave, convex,
irregular, or angular (Schaad et al., 2001). Phytopathogenic bacterialcells can be .
transparent, or produce pigments, the most common being yellow, white, and red. Other
diagnostically useful characteristics of plant pathogenic bacteria include, but are not
limited to, their ability to cause a hypersensitive response on tobacco, growth within
certain temperature ranges, motility, production of acid, and use of various nutrients
(Agrios et al., 2001).

Plant pathogenic bacteria reproduce and grow in different environments. They
live as epiphytes on the surface of plants, and are also able to inhabit the phloem or
xylem of plants. Phytopathogenic bacteria can also survive as saprophytes in dead or
decaying plant tissue (Hirano and Upper, 1983). Bacteria survive in soil, on seed, in the

digestive tracts of vertebrates and invertebrates, and in various water sources.

Plants can be infected with bacteria in different ways. Bacteria can be transferred
through the use of contaminated farm equipment, enter plants through the stomata, and
inﬁltrate plants through wounds caused by animals or weather (Hartman and Hayward,
1993). Another important way plants are infected with bacteria is by insects. Insects can
be vectors of bacterial plant pathogenic diseases by carrying bacteria on external body
parts or internally in the digestive system. For example, Erwinia amylovora (Burrill), a
bacterium which causes ﬁre blight in apple and pear trees, is carried on the body and legs
of honey bees (Apis mellifera), (Thomson et al., 1992). Honey bees transfer the
bacterium to the stigma of apple tree and pear tree ﬂowers when searching for nectar and
pollen in the ﬂowers. In contrast, blue-green sharpshooters (Graphocephala
atropunctata) are xylem feeding suctorial insects which carry Xylellafastidiosa in their
digestive systems (Purcell and Saunders, 1995). Xylella fastidiosa is the causal agent of
Pierce’s disease in grapevines. Sharpshooters transfer X. fastidiosa to grapevines by
feeding on grape clusters. Another important insect vector is the corn ﬂea beetle,
Chaetocnema pulicaria (Melsh), which when feeding on corn plants transfers Pantoea
stewartii causing Stewart’s bacterial wilt (Poos and Elliott, 1935; P005, 1955; Castor et
al., 1975; Adams and Los, 1986).

Definitions

The following vocabulary will be used throughout this thesis:
Pathogenicity. The ability for a pathogen to cause disease.
Phytopathogenic. The ability for a pathogen to cause disease in a plant.
Virulence. The relative capacity of a pathogen to cause disease in a host.

(more/less) Virulent. Degree of virulence exhibited by a pathogen on a host.

Avirulent. Lacking virulence.

Isolate. A pure culture of bacteria that was obtained by subculture of a single colony
from a common source (host).

Strain. An isolate that has been differentiated based on its (rep-PCR) genomic
ﬁngerprint pattern.

Clonal. Bacterial populations with little or no genetic change in the evolutionary history
of the species.

Panmictic. Bacterial populations with genetic changes that have happened frequently
during the evolutionary history of the species.
Stewart’s Bacterial Wilt

Stewart’s bacterial wilt is a disease of ﬁeld corn and sweet corn (Zea mays L.)
that is caused by the bacterium P. stewartii (Frutchey, 1936). The symptoms associated
with Stewart’s bacterial wilt include stunted plant growth, wilting of leaves, and linear
water soaked lesions on the leaf surface. Stewart’s bacterial wilt symptoms were ﬁrst
discovered on sweet corn in 1894 by EC. Stewart in New York. Stewart determined that
the cause of the symptoms was due to infection by a bacterium and sent a culture to
Erwin F. Smith for taxonomic analysis. In 1897, Smith named the bacterium
Pseudomonas stewarti, but over the years it has been renamed and is currently Pantoea
stewartii (Mergaert et al., 1993).

Pantoea stewartii is a non-motile, gram negative, facultatively anaerobic,
baccillus bacterium (McGee, 1998). On solid media, such as nutrient broth agar or Luria-
Bertani agar, the bacterial colonies produce a yellow pigment. The optimal growth is 25-
27 °C and it will not grow at temperatures of 37 °C or higher (Schaad et al., 2001). The

bacterium produces a positive hypersensitive response when injected into tobacco leaves.

Once infected into the corn plant, P. stewartii can spread systemically through the
vascular system and has been successfully extracted from leaves, stalks, roots, and seeds
(Adams and Los, 1986; Munkvold et al., 1996; Pataky et al., 2000). An
exopolysaccharide (EPS) may be responsible for its ability to colonize the vascular
system of sweet corn (Braun, 1990; Coplin etal., 1992; Leigh and Coplin, 1992; Denny,
1999). The EPS may act as an anchoring mechanism allowing bacteria to remain in areas
of the plants vascular system where nutrients are abundant, resulting in obstruction of
water and nutrient pathways causing the plant to wilt and die (Blanco et al., 1979; Leigh
and Coplin, 1992). Coplin et al. (1990) suggested that the EPS protects the bacterial cells
from plant defense systems. Researchers are also studying plasmids contained in the
bacterial cell for other pathogenicity factors (Coplin et al., 1992; Fu et al., 1996, 1997,
1998).

Taxonomy

In 1993, three members of the genus Erwinia were classiﬁed as belonging to the
genus Pantoea. Classiﬁcation of members of the genus Erwinia has been under much
scrutiny in the last ten years because of the diversity in morphology, genetic make-up,
and pathogenicity observed among species classiﬁed in this genus (Mergaert et al. 1993).
Morphological characteristics that are shared by members of the genus Erwinia include
being Gram negative, the ability to grow both anaerobically and aerobically, the inability
to grow at 37°C, and the lack of ability to produce spores (Schaad et al., 2001). The
characteristics that are not shared among all Erwinia genus members include bacterial

colonies possessing a yellow pigment when grown on certain media, the production and

utilization of pectate lyases and cellulases, and the symptoms produced on plants that
include blights, wilts, and soft-rots.

Researchers have worked to better understand, reorganize, and possibly disband
the genus Erwinia using molecular techniques that include DNA-DNA hybridization,
protein proﬁles, G+C content comparisons, and the sequencing of speciﬁc genomic
regions (Mergaert et al. 1993; Kwon et al., 1997). Utilizing molecular and morphological
results, three subgroups have been loosely deﬁned. Group one is the soft rot group which
contains the carotovora and chrysanthemi species (Kwon et al. 1997). This group has
been deﬁned by its ability to utilize pectate lyases and cellulases to degrade cell walls
causing soft rot and by the similarity in their G+C content (Mergaert et al. 1993). Group
two is the amylovora group that includes the amylovora, nigrrﬂuens, salicis,
rubrifacinens, and rhapontici species (Mergaert et al. 1993; Kwon et al., 1997). The
amylovora group contains bacteria that primarily infect trees and have been grouped
based on their carbon utilization and genomic structure (Kwon et al., 1997). The last
group that has historically been a part of the genus Erwinia, but is now referred to as the
genus Pantoea contains the species stewartii, ananas, and uredovora (Mergaert et al.
1993). Mergaert et al. (1993) separated these species to the genus Pantoea based on
DNA-DNA hybridization and protein proﬁles. Pantoea stewartii has been previously
classiﬁed as; Pseudomonas stewartii (1887 E. F. Smith); Phytomonas stewartii ( 1918
E.F. Smith); Bacillus stewartii (1920 E.F. Smith); Xanthomonas stewartii (1939 E.F.
Smith); Erwinia stewartii (1963 E.F. Smith); (Pepper, 1967; Mergaert et a1. 1993).

The separation and classiﬁcation of the Erwinia genus is still under much debate.

Kwon et al. ( 1997) attempted to resolve some of the ambiguity by analyzing the

relationships between 16 different Erwinia species based on their 16S rRNA gene
sequences. Six members of the family Enterobacteriaceae, of various genera, were also
included to help clarify the proposed hypothesis that the genus Erwinia is polyphyletic
(Kwon et al., 1997). The research performed by Kwon et al. (1997) used an evolutionary
distance relationship described by J ukes-Cantor in the form of a neighbor-joining tree to
analyze the sequences of the 168 rRNA in each of the 22 species. The conclusion of
Kwon et al. (1997) was that the Erwinia genus is polyphyletic. More recently, Durkin
(unpublished data 2001) reanalyzed the same 16S rRNA sequences using a maximum
parsimony character-state approach and a maximum likelihood analysis to show that a
polyphyletic relation between members of the Erwinia and Pantoea species was not
statistically more likely than a monophyletic relation. The classiﬁcation of the genera
Erwinia and Pantoea is currently still under debate.
The Vector: Chaetocnema pulicaria

The vector of P. stewarrii in corn plants is the corn ﬂea beetle, Chaetocnema
pulicaria (Poos and Elliott, 1935; Pepper, 1967; Adams and Los, 1986). The bacterium
is carried in the digestive system of the beetle and transferred to the corn plant when the
beetles feed on plant tissue (Poos and Elliott, 1935; Poos, 1955; Adams and Los, 1986).
Feeding usually occurs on the leaf surface where the adult beetle chews and removes a
superﬁcial layer of the leaf, but feeding on the roots by com ﬂea beetle larvae has also
been observed (Poos and Elliott, 1935; Poos, 1955; Adams and Los et al., 1986). The
beetles feed primarily on sweet corn, but have also been known to feed on crabgrass,
orchard grass, ryegrass, and fall panicum (Adams and Los, 1986). Because of their

minute size (2 mm long), damage caused to the leaf of a corn plant by single beetles is

minimal, wounds are 1 mm wide and 1-30 mm long, but large numbers of beetles on
individual plants have the ability to cause extensive structural damage (Poos and Elliott,
1935; Poos, 1955; Adams and Los, 1986). The economic impact from corn ﬂea beetle
infestation is the beetle’s ability to vector P. stewartii.

The life cycle of the corn ﬂea beetle has been studied to better understand and
predict when epidemics of Stewart’s bacterial wilt may occur. The corn ﬂea beetle over-
winters as an adult in the soil or plant debris and re-emerges in the early spring when
sweet corn is beginning to emerge (Adams and Los, 1986). Soon after beetles emerge
from the ground, they lay their eggs in the soil (Pepper 1967; Adams and Los, 1986).
These larvae emerge mid-summer. Adults can continue to transmit P. stewartii to
uninfected plants after feeding on infected plants (Adams and Los, 1986). In 1934, N. E.
Stevens ( 1934) developed a winter temperature index that predicted the over-winterin g
probability of corn ﬂea beetles. The index is applied by measuring the sum of the
average temperatures (°C) during December, January, and February. According to the
index, if the sum of the three months is less than 0.0°C then there is a low risk of
Stewart’s bacterial wilt, but if greater than 0.0°C then the risk of Stewart’s bacterial wilt
infection is high (Stevens, 1934). It has been shown that only 10-30% of over-wintering
adult beetles carry P. stewartii, but this winter temperature index makes the assumption
that all beetles are carriers of P. stewartii (Pepper, 1967). Stevens (1934) also developed
a threshold that estimated when beetle populations could be a viable crop problem. The
threshold was exceeded if 6 beetles were observed on 100 plants (Stevens, 1934). Other
reliable methods of beetle scouting and collection include the use of yellow sticky traps

and sweep nets (Adams and Los, 1986). The temperature and threshold indices are

presently the standard tools for predicting epidemics of Stewart’s bacterial wilt
throughout the eastern United States.
Stewart’s Bacterial Wilt Epidemics

Historically, the incidence of Stewart’s bacterial wilt in United States sweet corn
ﬁelds may be described as sporadic. After its discovery in New York by Stewart in 1894,
the incidence of Stewart’s bacterial wilt in the eastern United States was limited to a few
individual sweet corn plants until a severe outbreak occurred in 1932 (Frutchey, 1936).
During the 1930’s, researchers attempted to answer questions of how and where
inoculum levels of P. stewartii accumulated to levels that would cause such a severe
epidemic (Frutchey, 1936). Researchers directed their studies of P. stewartii control
towards survival on seed, transmission of bacterium by insects, and breeding for
resistance (Stevens, 1934; P003 and Elliott, 1935; Frutchey, 1936). It was concluded that
the ability of P. stewartii to survive on sweet corn seed was low, less than 3% of infected
seed produced plants with Stewart’s bacterial wilt symptoms (Frutchey, 1936). More
recent studies conﬁrmed that the chance of transmitting P. stewartii by seed was minimal
(< 5% infected seed produced by infected plants) based on seed infection studies (Block
et al., 1998, 1999). Entomological researchers concluded that beetle members of the
genus Chaetocnema, most importantly the corn ﬂea beetle (C. pulicaria), were the
primary vectors of P. stewartii (Poos and Elliott, 1935). Presently, the corn ﬂea beetle is
recognized as the primary vector and source of inoculum for Stewart’s bacterial wilt
infection (Munkvold et al. 1996). Lastly, Reddy and Holbert (1928) were able to achieve
limited control of Stewart’s bacterial wilt by testing inbred and hybrid varieties of sweet

corn that showed resistant to Stewart’s bacterial wilt (Frutchey, 1936). Patakay et al.

(2000) has shown that there are some varieties that are more resistant to Stewart’s
bacterial wilt infection, but even these varieties can still be killed if infected with high
populations (106-10" colony forming units (CFU) / ml) of P. stewartii.

After the Stewart’s bacterial wilt outbreak in 1932, the incidence of Stewart’s
bacterial wilt in the eastern United States was limited to the infection of a few individual
sweet corn plants even when high levels of corn ﬂea beetles were observed, until the late
1960’s and early 1970’s (Pepper, 1967; D. L. Coplin, pers. comm). During this time, the
incidence of Stewart’s bacterial wilt of sweet corn again reached epidemic proportion in
parts of the eastern United States (D. L. Coplin, pers. comm). High levels of corn ﬂea
beetles vectoring P. stewartii were again determined to be the cause, but there was no
explanation for the sudden increase in P. stewartii inoculum levels.

After the early 1970’s, Stewart’s bacterial wilt was again only observed
sporadically on a few individual plants throughout the eastern United States until another
epidemic occurred during the 1989 growing season in New York State, the 1990-1992
growing season in Iowa, the 1995 growing season in Illinois, and the 1996-98 growing
seasons in Michigan (Dillard and Kline, 1989; Pataky et al. 1996, Munkvold et al., 1996,
W. Kirk, pers. comm). At this time, researchers shifted away from corn ﬂea beetle
population studies and began to explore population structures of the bacterial pathogen,
P. stewartii. Wilson and Dillard, 1999, utilized the Biolog Inc. (Hayward, CA) Biolog
gram-negative microplate which contained 95 carbon sources to determine the
phenotypic variability of 124 P. stewartii isolates collected from 11 different states in the
Eastern United States ranging in collection dates between 1932-1994. It was determined

in this study that all of the isolates except one shared the ability to utilize 93% of the

same carbon sources suggesting limited diversity in the phenotype of the different
isolates (Wilson and Dillard, 1999). Wilson and Dillard (1999) hypothesized that limited
phenotypic diversity may correspond to a limited genotype diversity. Coplin et al. (2002)
utilized pulsed ﬁeld gel electrophoresis (PFGE) to separate restriction fragments
produced by two restriction endonucleases and determined that isolates from separate
regions could be differentiated at the strain level using this technique. Although it was
not the primary objective of their research, Coplin et al. (2002) concluded that the strains
analyzed seemed to have a low diversity, based on the large amount of shared restriction
fragments. Presently, the genetic population diversity of strains of P. stewartii is still
uncertain.
Bacterial Population Analysis

Populations of bacterial species have varying levels of genetic diversity (Smith et
al., 1993; Spratt and Maiden, 1999). Bacteria reproduce asexually by binary ﬁssion,
which consists of a mother cell giving rise to two identical daughter cells (Smith et al.,
1993). Genetic changes in the chromosome of bacterium most frequently occur through
point mutation or recombination by transformation, conjugation, or transduction. There
is evidence that recombination and mutation rates may vary broadly between different
bacterial populations (Spratt and Maiden, 1999). Therefore, bacterial population
structures can range from being clonal to panmictic. Most bacterial populations are
somewhere intermediate between the classiﬁcation of clonal and panmictic. Bacterial
populations have also been deﬁned as ‘epidemic clonal’, which is a panmictic population
that sporadically arises as a virulent strain for a short period of time, but survives as an

avirulent or less virulent organism over an extended duration. It is theorized that speciﬁc

10

events occur that initiate recombination in bacterial population structures resulting in a
pathogen population that is more virulent over a short period of time (Spratt and Maiden,
1999).

Random whole-genome analysis (RWGA), speciﬁcally repetitive-sequence-based
polymerase chain reaction (rep-PCR), can help to differentiate between members of a
bacterial population (Louws et al., 1994). The rep-PCR technique uses primer sets based
on conserved repetitive bacterial elements (De Bruijn, 1992; Louws et al., 1999). These
primers include the repetitive extragenic palindromic (REP) sequence (35-40 bp) and the
BOX element that is made up of boxA, boxB, and boxC (154 bp), (de Bruijn, 1992).
When these primers are used in the rep-PCR technique, they are. referred to as REP-PCR
and BOX-PCR (Louws et al., 1996). PCR ampliﬁcation by these primer sets can yield
genomic ﬁngerprints speciﬁc to individual strains of phytopathogenic bacteria. For
example, this technique has been used to differentiate within phytopathogenic strains of
Xanthomonas and Pseudomonas that were previously not distinguishable with other
classiﬁcation methods (Louws et al., 1994). Since phytopathogenic strains of
Xanthomonas and Pseudomonas have a wide range of hosts, rep-PCR was shown to be a
quick and effective tool which could differentiate these bacteria at the pathovar level.
The rep-PCR technique was also used by Louws et al. (1998) to differentiae between ﬁve
Clavibacter michiganensis subspecies that were pathogenic on different hosts (Louws et
al., 1998). They also found that rep-PCR could be used as a tool to differentiate between
members of the subspecies C. michiganensis subsp. michiganensis. This subspecies was
divided into ﬁve rep-PCR strain types based on genomic ﬁngerprint polymorphisms.

Although a biological explanation was not concluded, the rep-PCR strain type groupings

11

also corresponded to strain groupings based on their known virulence (Louws et al.,
1998). More recently, Horita and Tsuchiya (2000) assessed the genetic diversity of 74
strains of Ralstonia solanacearum, which has an extremely wide host range, and were
able to determine that although strains were morphologically similar, they were easily
separated into races based on their rep—PCR genomic ﬁngerprints.
Objectives of this Research

This research incorporates objectives that will assess both genetic variability and
virulence of a range of isolates of P. stewartii from the eastern United States. Deﬁning
the genetic variability of P. stewartii isolates from different temporal and geographic
locations may offer Stewart’s bacterial wilt researchers an epidemiological tool that can
be used when epidemics occur in the future. Also, the development of a procedure that
can assess if variation in virulence exists between different isolates of P. stewartii may be
used as a tool to differentiate P. stewartii strain populations that are present during an
epidemic.
In order to fulﬁll these objectives three procedures must be performed:
Speciﬁcally;

1) Collect P. stewartii isolates that represent different geographical locations and

different time periods in the eastern United States by:

a) contacting researchers of Stewart’s bacterial wilt from the eastern
United States who maintain P. stewartii collections.

b) capturing and assaying corn ﬂea beetles for P. stewartii from Michigan
sweet corn ﬁelds

c) placing a susceptible variety of sweet corn in Michigan sweet corn
ﬁelds as bait

12

d) scouting sweet corn ﬁelds in Michigan for the presence of Stewart’s
bacterial wilt infection

2) Determine if the rep-PCR technique can be used to differentiate between
members of a closely related genus (Erwinia and Pantoea) as well as
differentiate between isolates of the same species (P. stewartir), and if so use
BOX-PCR and REP-PCR as a tool to determine if variability exists between
P. stewartii isolates.

3) If strains of P. stewartii can be differentiated using the rep-PCR protocol,
determine the variability in virulence between different strains of P. stewartii

on sweet 00m.

13

CHAPTER 2

MATERIALS AND METHODS

2.1 Isolates Studied. Eighty-four P. stewartii isolates were obtained from both the 2001
growing season and from culture collections (Table 1). Three of the isolates were
assayed from Michigan (PS-II) sweet corn during the 2001 growing season. Fourteen of
the isolates assayed (Ps-V; VI; VII) were from sweet corn in three regions of Illinois
during the 2001 growing season. Twenty-ﬁve isolates (SW 87) were obtained from
sweet corn in Iowa and stored by Greg Larnka in 1987 (Lamka, 1991). Twenty-nine
isolates (ES 92) were obtained from sweet corn in Iowa and stored by Charles Block in
1992. Fourteen isolates were obtained from Dr. D. Coplin’s culture collection at Ohio
State University through Dr. C. Block. Two isolates were used as a positive controls:
Pantoea stewartii — ATCC-29227 (Margaert, 1993) which was obtained from the
American Type Culture Collection (ATCC) and Il-Ps 1 which was obtained from Dr. J.
Pataky of the University of Illinois, Urbana.
2.2 Michigan Isolate Collection Techniques. In 2000, P. stewartii was not found in
sweet corn plants or corn ﬂea beetles and there were no reports of Stewart’s bacterial wilt
infection by Michigan sweet corn growers. Therefore, Michigan isolates were collected
only over the course of the 2001 growing season (May 15 - Aug 25). Twelve commercial
sweet corn ﬁelds, ranging in size from 15 to 25 ha, located in the southern half of the
Lower Peninsula in Michigan were surveyed on a ten day schedule starting May 15 for
Stewart’s bacterial wilt infection (Figure 1).

Three techniques were used to ﬁnd isolates of Stewart’s bacterial wilt. Scouting;

15 different rows 100 m long were inspected. All sweet corn plants that exhibited any

14

Table 1. Bacterial isolates used in this research for rep-PCR analysis and virulence

differentiation on sweet corn.

 

 

Presence (+) Presence (4)
. Absence (-) Absence (-) PCR
Genus- . a b Location of 700 bp of 380 bp band .
. Strain Source c . . ﬁngerprint
Specres Source band In In t a
(BOX-PCR) (REP-PCR) YP"
ﬁngerprint ﬁngerprint
Pantoea ATCC -29227 ATCC United States + + 1
stewartu
University of
ll-Ps lc SCP Illinois, Urbana - + 3
Dr. Pataky
Riley Center, Ml,
Ps-ll-B-CP-l SCP (2001) - + 3
Riley Center, M1,
Ps-ll-B-CP-3 SCP (2001) - + 3
Riley Center, Ml.
Ps-ll-B-CP-4 SCP (2001) - + 3
Champaign, IL-
Ps-V-A-CP- 1 -1 SCP Dr. Pataky Lab - + 3
(2001)
Champaign, IL-
Ps-V-A-CP-l-Z SCP (2001) - + 3
Champaign, IL-
Ps-V-A-CP-3-l SCP (2001) - + 3
_ - _ _ _ Champaign, IL-
Ps v A C? 3 2 SCP (2001) - + 3
_ _ _ _ Champaign, IL-
Ps v A CP-4 1 SCP (2001) - + 3
- _ Champaign, IL-
Ps-V-A CM 2 SCP (2001) - + 3
Mendota, IL
Ps-Vl-A-CP-l-l SC? (2001) - + 3
Mendota, IL
Ps-Vl-A-CP-l-Z SCP (2001) - + 3
Mendota, IL
Ps-Vl-A-CP-l-B SCP (2001) - + 3

 

15

 

 

Presence (+) Presence (+)
Loc ' Absence (-) Absence (-) PCR
- . atron .
Genus Strarn' S on r C en c of 700.bp of 380.bp band ﬁngerpnm
Specres Source band In In t d
(BOX-PCR) (REP-PCR) YPC
ﬁngerprint ﬁngemrint
Pantoea. Ps-VI-A-CP-Z-l S CP Mendota, IL _ + 3
stewartu (2001)
Mendota, IL
Ps-Vl-A-CP-2-3 SCP (2001) - + 3
Mendota, IL
Ps-Vl-A-CP-4-l SCP (2001) - + 3
Dixon Springs, IL
Ps-Vlll-A-CP-3-l SCP (2001) - + 3
Dixon Springs, IL
Ps-Vlll-A-CP-3-2 SCP (2001) - + 3
OSU
SW ‘ SCP (Ohio) (1974) + ‘ 2
OSU
SW 2 SCP (Ohio) (1974) ‘1' ' 2
OSU
3‘" ‘3 SCP (Indiana) (1974) + + 1
OSU
sw14 SCP (Indiana) (1974) + + 1
OSU
SW ’9 SCP (Kentucky) (1975) + ‘1’ 1
OSU
SW 36 SCP (Tennessee) (1975) + + 1
OSU
SW 39 SCP (North Carolina) + + 1
(1975)
OSU
SW 45 SCP Illinois (1975) + + 1
OSU
MU 51 SCP (acapsular virulent + - 2
mutant)
OSU
GAL E8 SCP (spontaneous + - 2
avirulent mutant)

 

16

 

 

Presence (+) Presence (+)
, Absence (-) Absence (-)
Genus- - a b Locatwﬂ of 700 bp of 380 bp PCR -
. Strain Source c b d . b d . ﬁngerprint
Specres Source an m an tn d
(BOX-PCR) (REP-PCR) tYPed
ﬁngerprint ﬁngerprint
DC 160 scp 03” + - 2
(New York) (1976)
OSU
DC 283 SCP (spontaneous + - 2
88104- mutant)
OSU
~ (rifampicin resistant _
le 98 SCP strain-derived from + 2
DC 283)
SW 87-1 -SW 87-5
SW 87-7 - SW 87-13 ISU
sw 87-15-16; 18 SCP (1987) "' "' 1
SW 87-20-23; 25; 26
_ . _ ISU
SW 87-6. 17. 19. 24 SCP (1987) + - 2
ES 9201 SCP Washington, IA + + 1
ES 9203 SCP Lonetree Iowa + + 1
ES 9204 SCP Washington Iowa + + 1
ES 9205 SCP Nevada, Iowa + + 1
ES 9206 SCP Nevada, Iowa - + 3
ES 9207 SCP Story City Iowa + + 1
ES 9208 SCP Boone, Iowa + + 1
ES 9209 SCP Boone, Iowa + + 1
ES 9210 SCP Boone, Iowa + + 1
ES 9211 SCP Slater, Iowa + + 1
ES 9212 SCP Story City Iowa + - 2
ES 9213 SCP Slater, Iowa - + 3
ES 9221 SCP Washington, IA 4» + 1

 

17

 

 

Presence (+) Presence (+)
_ Absence (-) Absence (-) PCR
Genus- . a b Location of 700 bp of 380 bp .
. Strain Source c band. b d. ﬁngerprint
Specres Source "1 an In t o
(BOX-PCR) (REP-PCR) ype
ﬁngerprint ﬁngerprint
Pantoea
stewartii ES 9225 SCP Washington, 1A + + 1
ES 9222 SCP Washington, 1A + + 1
ES 9230 SCP Maxwell, IA + - 2
ES 9243 SCP Slater, Iowa + + 1
ES 9244 SCP Slater, Iowa + + 1
ES 9245 SCP Nevada, Iowa - - 4
ES 9246 SCP Coon Rapids, IA + + 1
ES 9247 SCP Slater, Iowa + + 1
ES 9248 SCP Madrid, IA + + 1
ES 9249 SCP Story City Iowa + + 1
ES 9250 SCP Madrid. IA + + 1
ES 9253 SCP Schaller, IA + + 1
ES 9256 SCP Coon Rapids, IA + - 2
ss 104 ICPB Illinois (1967) + - 2
ES 9257 SCP Grundy Center, IA - + 3
OSU f
P. anannas DC 130 SCP New York na na na
aimcga 10 AT Michigan na na na
321 France na na na
MRI R Michigan na na ’ na

 

18

 

Presence (+) Presence (+)

 

Genus- . Location Absence H _ Absence H . PCR.
. Straina Sourceb c of 700 bp band In of 380 bp band In ﬁngerprint
SpCCICS SOUI‘CC (BOX-PCR) (REP-PCR) typed
ﬁnge_rprint ﬁnge_rprint

E. amylovora PSLN Mlchlgan na na na
CFB Pr uk uk na na na
E. carotovora ng uk uk na na na
1'1 chrysrhami uk uk uk na na na

 

I'The designation given to each strain. For Michigan and Illinois strains Es=Erwinia stewartii,
Ps=Pantoea stewartii the roman-numeral=the region, the letter = the ﬁeld, the CP=potted sweet
corn plant, and the number indicates the order in which it was collected. For the Iowa strains the
BS or the SW = Pantoea stewartii and Stewart’s Wilt respectively. The ﬁrst two digits equal the
year and the last two digits indicate the order in which they were collected.

-Strains Ps-II- Matthew Durkin, Michigan State University

-Strains Ps-V, VI, VIII - Dr. J Pataky University of Illinois, Urbana

-Strains Es 92 - Dr. Charles Block’s Culture Collection, Iowa State University

-Strains SW 87 - Dr. Greg Lamka’s Culture Collection, Iowa State University

-Strains from OSU- Dr. D. Coplin Culture Collection, Ohio State University

-E. amylovora strains - Dr. A1 Jones Culture Collection, Michigan State University
b Source: ATCC - American Type Culture Collection; SCP - Sweet Corn Plant Tissue; ICPB —
International Collection of Phytopathogenic Bacteria, University of California, Davis; AT --
Apple Tree; R — Raspberry.
° City, state, or country where the isolate was sampled from (if known).
d Designation given to a strain based on its genomic ﬁngerprints from rep-PCR using the BOX
and REP primers.
‘ Population of strains maintained on greenhouse sweet corn plants. The population originated 10
to 15 years ago and was assayed from a sweet corn plant collected in Urbana, Illinois by Dr. J
Pataky. The population has been added to each year with populations from infected leaf tissue
collected in Urbana, Illinois.
’ Not applicable.
3 Unknown.

19

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

“N-.___

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1. Southern portion of Michigan’s Lower Peninsula showing the twelve
locations where isolates of Pantoea stewartii were sought. The roman numerals
represent the regions and the lettered stars represent the closest city to the ﬁelds.
I-A-Fremont; I-B-Grand Rapids; I-C-Byron City; II-A-Romeo; H-B-Almont;
II-C-Riley Center; III-A-Chelsea (ﬁeld 1) III-B-Chelsea (ﬁeld 2); III-C-Monroe;
IV-A-Dowagiac; IV-B-Decatur; IV-C-Marcellus.

20

evidence of Stewart’s bacterial wilt in the grower’s ﬁelds were harvested and assayed for
P. stewartii. Bait; 25 potted (pot size = 20.5 cm) Jubilee sweet corn plants were placed as
bait in weeds around the margins of the ﬁelds until mid-June, after the grower’s sweet
corn reached the 3-5 leaf stage, when they were then placed between sweet corn plants in
the field. The pots were placed in ﬁve separate rows, ﬁve pots per row with 5 m spaces
between each pot. Plants with symptoms of Stewart’s bacterial wilt or corn ﬂea beetle
feeding scars (1-30 mm lines 1 mm wide where the epidermis had been removed on the
leaf tissue) were assayed for P. stewartii. Sweeping; corn ﬂea beetles were captured with
an insect sweep net (38 cm diameter rim and polyester net bag). Four sweeps were
performed per visit in the sweet corn ﬁeld and in the weeds that surrounded the ﬁeld.
Single sweeping events were conducted over 100 In sweeping the net approximately 150
times. The contents of the net bag were emptied into a l L plastic Ziploc® bag from
which corn ﬂea beetles were removed, identiﬁed, and assayed (described in section 2.3)
for P. stewartii.

2.3 Isolation Techniques.

2.3.1 Sweet Corn Plants. Each sweet corn sample that was suspected to be infected with
Stewart’s bacterial wilt was assayed for the presence of P. stewartii by removing a 1.5
cm2 section of leaf tissue from a symptomatic sweet corn sample. The section was placed
in 0.4 ml of buffered saline (0.01 M potassium phosphate, 0.8% sodium chloride, pH 7.0)
in a sterile porcelain mortar and macerated using a sterile porcelain pestle for 10 seconds

(Ivanoff, 1933). The macerated solution was then streaked using a 1 p1 sterile loop on a

plate (100 x 15 mm) containing nutrient broth agar (1.5% agar, 8 g nutrient broth/ L

21

Difco-NBA). The plates were incubated in the dark for 48 hours at 23°C. Single
colonies that had the general characteristics of P. stewartii (yellow pigment, < 3 mm in
size) were picked and re-streaked on NBA plates. The procedure was repeated 3
consecutive times to ensure the purity of the culture so a frozen stock sample could be
preserved. A frozen stock sample was prepared by removing a single colony from the
ﬁnal culture, which was transferred to a 12 x 150 mm screw cap culture tube containing
5 ml of nutrient agar, and incubated for 24 hours at 23 °C on a shaker at 200 rpm. A 1.0
ml sample of the solution was placed in 0.5 ml of 50% glycerol and stored in 1.5 ml

centrifuge tubes at —80 0C in a freezer.

2.3.2 Corn Flea Beetle. Corn ﬂea beetles were macerated and assayed according to the
previous method (Section 2.3.1). The beetles were ﬁrst placed in a -20 °C freezer for 10
minutes to slow their activity. Each beetle was evaluated individually.

2.4 rep-PCR differentiation between members of the genus Erwinia and genus
Pantoea. Since it was not known if rep-PCR could differentiate between members of the
genera Pantoea and Erwinia (previously in the same genus) (Mergaert et al., 1993), a
preliminary rep-PCR run using the BOX primer (Versalovie et al., 1994) was completed.
Five strains of Erwinia amylovora (10, 321 , MRI, PSLN, CFB Pr), one strain of Erwinia
chrysamthemi, one strain of Erwinia carotovora, and two strains of P. stewartii [ATCC
29227 and Illinois control-(Il-Ps 1)] were ampliﬁed (Table 1). The BOX-PCR procedure
was performed as described by Versalovic et al. (1994) with some adjustments; mineral
oil was not added to all rep-PCR reactions because the thermal cycler used (GeneMate

Magnum Techne, Princeton, NJ, USA) had a heated hood that prevented evaporation; the

22

BOX primer was synthesized by IDT-Integrated DNA Technologies, INC, Coralville, IA,
USA; the electrophoresis procedure followed methods from Louws et al. (1998).

2.5 Identiﬁcation of Isolates (Gram Stain, Pathogenicity, BOX-PCR). Samples were

identiﬁed as P. stewartii isolates by Gram stain, pathogenicity on a susceptible variety of
sweet corn (Jubilee), and comparison of genomic ﬁngerprints using BOX-PCR.

The modiﬁed Gram stain protocol was performed as described by Schaad et al.
(2001). Bacterial samples were prepared by streaking single colonies on a NBA (8 g/L,
1.5% agar) plate (100 x 15 mm) and incubating them in the dark for 72 hours. A single
colony was transferred to a sterile screw cap vial (15 x 60 mm) containing 10 ml of
nutrient broth and incubated for 24 hours at 23 °C on a shaker at 200 rpm.

The pathogenicity of each isolate was determined by inoculating Jubilee sweet
corn plants at the 2-3 leaf stage (Figure 2) and assessing the potential of the bacterium to
cause Stewart’s bacterial wilt (Blanco et al., 1977). The sweet corn was sown in 20.5 cm
black plastic pots and grown in a greenhouse with an average temperature of 29 °C (i 3.2
°C), 14 hours daylight, 80% (1 10.2%) relative humidity. Inoculum was prepared by
streaking frozen stock samples of isolates on NBA plates and incubating at 23 °C for 48
hours. A single colony was transferred from the NBA culture to 5 ml of sterile nutrient
broth in a sterile screw cap vial (15 x 60 mm). The vial was incubated at 23 °C on a
shaker for 24 hours at 200 rpm. The absorbance (A6mnm) of each inoculation isolate was
between 0.39 and 0.41 (approximately 1 x 108 cfu/ml). In all experiments, inoculum
concentrations were determined by standard diluting and plating techniques. The plants
were inoculated using a 5 ml syringe with a 30.5 gauge needle. The ﬁrst step in the

inoculation technique was to load 1.0 ml of inoculum solution into the syringe. The

23

 

I" '

T‘U.

Leaf 1 ——> W5: -A

_‘

1:.“

' ‘ A:
."A

 

 

Soil Line

Figure 2. Sweet corn plant at the 2-3-leaf growth stage. The needle was inserted at the
arrow marked by the coleoptile label.

24

syringe plunger was then depressed to initiate ﬂow of inoculum prior to injection of the
plant tissue. The stem was then injected through the coleoptile sheath (approximately 1
cm from the soil surface) four times in 2 seconds while the inoculum was ﬂowing from
the needle.

The BOX-PCR procedure was used as a tool to construct genetic ﬁngerprints of
each isolate (protocol described in section 2.6).

2.6 rep-PCR using BOX-PCR and REP-PCR. The rep-PCR procedure, using the
BOX primer, was performed as described by Versalovic et al. (1994) with slight
adjustments. The amount of H20, per BOX-PCR reaction, was 13.34 pl and the amount
of primer was 1.16 pl (0.3 pglpl). Preliminary trials were performed to determine the
optimal BOX-PCR reaction for P. stewartii (example can be seen in Appendix A, Figure
15).

The rep-PCR procedure, using the repetitive extragenic palindromic (REP)
primer, was performed as described by Versalovic et al. (1994) with slight adjustments.
The amount of H20, per REP-PCR reaction, was 13.64 pl. Preliminary trials were
performed to determine the optimal REP-PCR reaction for P. stewartii (example can be
seen in Appendix B, Figure 16). The REP primer was synthesized by IDT-Integrated
DNA Technologies, INC, Coralville, IA, USA.

The electrophoresis procedure followed methods from Louws et al. (1998). The
ethidium bromide stained gels for the rep-PCR reactions (BOX-PCR and REP-PCR)
were photographed with a Polaroid camera and Type 57 High Speed Polaroid Film
(Polaroid Corporation, Cambridge, MA, USA). The photographs were transferred to

computer images using a Hewlett-Packard Scanjet 4C digital scanner (Hewlett-Packard

25

Company, Palo Alto, CA, USA.) and saved as TIFF ﬁles. Images were enhanced by
inverting them and adjusting the brightness and contrast using Adobe Photoshop 6.0
(Adobe Systems Incorporated, San Jose, CA, USA). Gel ﬁngerprints were analyzed
based on the presence or absence of fragments between the size of 350 base pairs (bp)
and 3000 bp for each isolate.

Genetic fingerprints of the 84 isolates of P. stewartii were produced using both
BOX-PCR and REP-PCR. BOX-PCR and REP-PCR were repeated four times on each of
the isolates. Bands were considered to be present if they were observed in all four of the
ﬁngerprint patterns. A pairwise similarity matrix was constructed using p-distances by
ﬁrst determining the proportion (p) of bands differing between any particular pair of
BOX-PCR or REP-PCR ﬁngerprint patterns. The equation for p-distance used was;

It

_ d
p_—
n

where nd = the number of differences, and n = the total number of bands. Distances were
generated using PAUP* v 4.0b4a (Swofford, 2002), and an index of similarity (s) was

then calculated using the equation;

where d = p-distance.

2.7 Virulence Assessment Protocol (Leaf Symptom Analysis). Four varieties of sweet
corn that differed in susceptibility to Stewart’s bacterial wilt from susceptible to resistant
(Table 2) were inoculated with ﬁfteen P. stewartii strains (Table 3), as described above.
The varieties of sweet corn were chosen based on ratings performed by Pataky et a1.

(2000). The sweet corn varieties included two resistant, one moderate, and one

26

Table 2. Varieties of sweet corn used in virulence assessment protocol.

 

 

Variety SW-ratlng' Kernel color Endosperm type Seed Source
Jubilee 9 yellow sub Rogers
Native Gem 5 bi-color sec Mesa-Maize
Buckeye 2 bi-color se Mesa-Maize
Ambrosia 1 bi-color se Crookham

 

" The susceptibility rating of the sweet corn variety to Stewart’s bacterial wilt infection.
The rating scale was developed by Dr. Jerald K. Pataky, Department of Crop Sciences,
University of Illinois, Urbana, IL, 61801 (Pataky, 2000). The rating scale is from 0-9
(0=resistant and 9=susceptible).

b Sugary.

° Sugary enhanced.

27

Table 3. Pantoea stewartii strain types used as inoculum in virulence assessment
protocol.

 

P. stewartil“
strain

 

Negative Control
ATCC-29227
Es 9211

Es 9208

SW 45

SW 87-2
SS104

Es 9230

SW 87-6

SW 87-19
SW 1

ll Ps-1
ll-B-CP-1
V-A-CP-1-1
VI-A-CP-1-1
ES 9245

 

aStrain information can be seen in Table 1.

28

susceptible to Stewart’s bacterial wilt (Table 2). The sweet corn was sown as described
in section 2.5. Four replicates of each treatment were positioned on top of 1 m high
benches in a random complete block design (RCBD). The experiment was performed
twice; starting dates were 8 Feb and 12 Feb, 2002. The P. stewartii strains were selected
based on strain types deﬁned by the rep-PCR protocol. The isolates were incubated on
NBA (8 g/ 1L, 1.5% agar) plates (100 x 15 mm) for 72 hours, and a single colony was
transferred to a sterile screw cap vial (15 x 60 mm) containing 10 ml of nutrient broth.
The vials were incubated at 23°C on a shaker for 24 hours at 200 rpm. The absorbance of
the inoculum was between 0.39-0.41 (approximately 1 x 108 chml) and was determined
based on the optical density at 600 nm. The plants were inoculated, as described in
section 2.5, 12 days after planting.

Forty-eight hours after inoculation, each plant was visually assessed for signs or
symptoms of infection (bacterial ooze, water soaked lesions, wilting, yellow streaking).
The ﬁrst, second, and third leaf were rated based on a 0-3 scale (O-no signs or symptoms;
1- slight loss of rigidity in leaf, water soaked lesions less than 1 cm in length; 2- drooping
of leaf, slight loss of rigidity in leaf, water soaked lesions greater then 1cm in length but
covering less than 50% of plant surface; 3- bacterial ooze, drooping of leaf, water soaked
lesions cover more than 50% of plant surface, general necrosis). Each leaf, 1, 2, 3 (Figure
2), was evaluated on each replicate plant every 24 hours for four days after ﬁrst
assessment.

2.8 Virulence Assessment Protocol (Colony Forming Units in Second Leaf Analysis).
Six days after inoculation, infection was quantitatively assessed based on the number of

colony forming units (CFU) recovered from the second leaf blade. The amount of

29

bacteria was determined by removing three (7 mm diameter) leaf disks from the second
leaf blade of individual plants six days after inoculation. The leaf disks were taken from
the base, middle, and tip of the leaf. The leaf disks were placed in a 1.5 ml centrifuge
tube with sterile buffered saline (0.3 ml, 0.01 M potassium phosphate, 0.8% sodium
chloride, pH 7.0). The leaf disks were macerated for approximately 10 seconds using a
tissue grinder. An aliquot (0.2 ml) of the ‘saline/leaf disk’ solution was removed from
the centrifuge tube and placed in a single well of a 96 well cell culture cluster (Corning
Incorporated 3593). The aliquot was serially diluted to 10‘1 by transferring 0.02 ml of the
‘saline/leaf disk’ solution to a well containing 0.18 ml of sterile buffered saline. The
process was repeated seven times, producing a 10" dilution, by transferring each newly
diluted solution to separate wells containing 0.18 ml of sterile buffered saline. After the
dilution was complete, 0.1 m1 of the 10'7 dilution solution was evenly spread on a NBA (8
g/ 1.0 L, 1.5% agar) plate (100 x 15 mm) for a ﬁnal dilution of 10". The plate was
incubated for 48 hours at 23°C. CFU on each plate that had the morphology of P.
stewartii were then counted and recorded. Colonies were conﬁrmed as P. stewartii by
randomly selecting 15 NBA plates that contained each of the 15 different bacterial
isolates previously inoculated into the sweet corn plants. The bacterial isolates were
positively identiﬁed as the original strains inoculated in the sweet corn plant by
comparing their genomic ﬁngerprints produced by using the BOX-PCR technique
(Appendix C, Figure 17).

2.9 Data analysis (Leaf Symptom Analysis). Statistical analyses were performed
using SigmaStat statistical software package (SPSS Inc., Chicago, IL, USA.) The area

under the disease progress curve (AUDPC) data were analyzed by two-way analysis of

30

variance (ANOVA) and means compared at p = 0.05 level of signiﬁcance by a multiple
range comparison of means (Tukey, SigmaStat). The AUDPC was calculated by adding
the area under the linear progression of symptom ratings (rating scale section 2.7)
between each successive rating of symptom severity from two days after inoculation to
six days after inoculation.

The AUDPC was estimated using the formula

where; n was the number of ratings, T was the time in days since inoculation and D was
the disease symptom rating (Campbell and Madden, 1990). The experiment was
performed two separate times and the data were combined because the variation between
treatments in the two experiments was not different. The AUDPC was calculated on; leaf
1 (AUDPCU), leaf 2 (AUDPCu), leaf 3 (AUDPCU), and for combinations of leaves; leaf
1,2,3 (AUDPCLH), leaf 2, 3 (AUDPCm). '

2.9.1 Data analysis (Colony Forming Units in Second Leaf Analysis). Statistical
analyses were performed using the Si gmaStat statistical software package (SPSS Inc.,
Chicago, IL, USA.) The number of colony forming units per leaf sample was not
normally distributed so the data were normalized by a’logio transformation. A two-way
analysis of variance was performed. The experiment was performed two separate times
and the data were combined because the variation between treatments in the two

experiments was not different.

31

CHAPTER 3

RESULTS

3.1 Michigan isolate collection. Over the course of the 2001 summer, a collection of
137 yellow-pigmented bacterial colonies were isolated and stored in frozen stock from
corn ﬂea beetles, potted susceptible varieties, and growers’ sweet corn from the 12 sweet
corn ﬁelds in the southern half of the Lower Peninsula of Michigan (Figure 1; Table 4).
Of the 137 colonies, three were found to be strains of P. stewartii based on Gram
staining, pathogenicity tests on Jubilee, and BOX-PCR genetic ﬁngerprinting. These
three isolates were assayed from different pots containing a susceptible variety of sweet
corn (Jubilee), but were found in the same ﬁeld, region II ﬁeld B (Figure 1). No other
isolates were identiﬁed as P. stewartii during the 2001 growing season and there were no
reports of infection in Michigan sweet corn ﬁelds.

3.2 rep-PCR (BOX-PCR) differentiation between members of the genus Erwinia
and genus Pantoea. The genetic ﬁngerprint of each Erwinia and Pantoea sample was
unique, and the differences found among members of the same species were minimal
(Figure 3). Each of the eight isolates contained 18-25 bands between the 350 to 4000-bp
range, but very few bands were shared among species. Among the two isolates of P.
stewartii, there were multiple bands that were the same size, but there was also a
polymorphism that differentiated the two isolates (ATCC-29227 and Il-Ps 1). Based on
the genomic ﬁngerprints obtained by using the rep-PCR procedure, it was determined
that using the rep-PCR procedure could differentiate between closely related Pantoea and

Erwinia species as well as within species of P. stewartii.

32

Table 4. Pantoea stewartii isolates collected from Michigan during the 2001 growing
season.

 

Total

 

 

number of Specimens 822:2; Gram-stain Pathogencit’y rep-PCR (BOX)
specimens Assayed" I c negative on Jubilee Identification“
. solated
inspected“
Scouting of 6
Growers ca. 1 x 10 30 12 12 0 0
Field'
Bait (Potted ‘
Corn Flea
Beetle ca. 480 320 121 95 0 0
Sweep“
Total - 355 137 1 1 1 3 3

" Scouting — 15 rows, 100 In, plants were spaced approximately 15 cm, 10 inspections
during the 2001 growing.
Baiting - 25 potted plants per inspection, 10 pct replacements during the 2001 growing
season.
Sweeping- 4 sweeps per inspection. 10 inspections during the 2001 growing season.

b The number of visually infected sweet corn plants from grower’s Michigan sweet corn
ﬁelds, Jubilee sweet corn plants that were placed in ﬁelds as bait and were visually
infected, and corn ﬂea beetles that were captured during sweeps.

Number of bacterial colonies (yellow pigment, < 3 mm in diameter) extracted from

' assayed specimens onto nutrient broth agar (NBA).

d Number of isolates that were able to cause symptoms of Stewart’s bacterial wilt when

inoculated into the sweet corn variety Jubilee.

Number of isolates with genomic ﬁngerprints similar to ATCC-29227 and Il-Esl using

BOX-PCR.

Scouting of ﬁelds consisted of walking through sweet corn ﬁelds and looking for signs

or symptoms of Stewart’s bacterial wilt on the sweet corn.

Potted Jubilee sweet corn plants (susceptible to Stewart’s bacterial wilt) were placed

around the margins and in sweet corn ﬁelds.

h Corn ﬂea beetles were captured in sweet corn ﬁelds using an insect sweep net.

0

‘5

33

 

 
 
   

650 bp
517 bp—>_

19999999991

Figure 3. BOX-PCR generated genomic ﬁngerprint, on a 1.5% agarose gel, of Pantoea
and Erwinia species. The numbered brackets below indicate the isolate of Pantoea or
Erwinia that is being analyzed. Each strain is repeated in adjacent lanes and
represented by the same number. The two lanes labeled with an ‘L' contain a 1 Kb DNA
Ladder. 2-Erwinia chrysanthemf, 3-Erwinia carotovora; 4-Erwinia amylovora (Es 110); 5-
E. amylovora (Es 321); 6-E. amylovora (Es MR1); 7-E. amylovora (PSLN); 8-E.
amylovora (CFB Pr). Lanes, 9-Pantoea stewartii (ll-Ps 1) and 10-Pantoea stewartii
(ATCC-29227) are differentiated by a 650 bp band that is absent in lane 9, but present in
lane 10.

34

3.3 BOX-PCR genetic variation of P. stewartii strains. When the BOX primer was
used in the rep-PCR procedure, unique genomic ﬁngerprints were found between the 84
isolates of P. stewartii which were collected from different geographical locations
(Appendix D, Figures 18-33). Each isolate shared 18-19 fragments between the 350 to
4000-bp range. Only one band (approximately 650-bp in size) differentiated the
collection based on its absence or presence in each of the 84 isolates. Examples of the
BOX-PCR can be seen in Figure 4. The 650-bp band was absent in all isolates collected
from Michigan and Illinois during the 2001 growing season and were similar to three
isolates collected from Iowa in 1992 by Dr. Charles Block (Es 9206; 45; 57). Two of
these isolates were from the same city (Nevada, Iowa), but from different ﬁelds. All
other isolates in this study had the 650-bp fragment.

3.4 REP-PCR genetic variation of P. stewartii strains. REP-PCR was used to further
differentiate the 84 strains of P. stewartii based on their genomic ﬁngerprints in
combination with the BOX-PCR ﬁngerprints (Appendix E, Figure 34-50). Each isolate
shared 16-17 fragments between the 350 to 4000-bp range (Figure 5). Like BOX-PCR,
only one band (approximately 380-bp in size) differentiated the isolates based on its
absence or presence in each of the 84 isolates. The 380-bp fragment was absent from
isolates collected from Ohio in 1974 (SW 1, SW 2), SS 104 (Illinois-1967), DC 160 (New
York-1976), DC 283 (spontaneous mutant of S8104), MU 51 (capsular avirulent mutant),
GAL E8 (EPS spontaneous mutant), four of the 25 isolates collected by Greg Lamka in
1987 (SW 87-6; 17; 19; 24), and two of the 28 isolates collected by Dr. Charles Block in

1992 (Es 9212, Es 9256). All other isolates in this study had the 380-bp fragment.

35

  
  

1018 bp—> ......

517 bp—>

 

I .‘m m

Figure 4. BOX-PCR generated genomic fingerprint, on a 1.5% agarose gel, of 15
Pantoea stewartii strains that were used in the virulence trial. The numbered lines
with arrows below indicate the isolate of P. stewartii that is being analyzed. The
three lanes labeled with an ‘L' contain a 1 Kb DNA Ladder. Strains missing the 650-
bp band - Lane 2-lllinois (Ps-Vl-A-CP-1-1); 3-lllinois (Ps-V-A-CP-1-1); 13-lowa (Es
9245); 15- Michigan (Ps-ll-B-CP-1); 16- Illinois (ll-Ps 1). Strains with the 650-bp
band- Lane 4-lowa (SW 87-2); 5-lllinois (SW 45); 6-Ohio (SW 1); 7-lowa (SW 87-
19); 8-Iowa (SW 87-6); 10-lowa (Es 9208); 11-lowa (Es 9211); 12-lowa ( Es 9230);
14-Illinois (SS 104); 17-ATCC-29227

36

   
   

1636-bp—> -‘au ﬁg

1013'bP-> - tinnitus-“ﬁlms“ .
“a, u'nv k-t-ri 'W 4&1 '
up” in. am;

.93.- «a

m , i t Q'— 380-bp

11:1: 1 z 1 i H T T H? 'T

1011121314151617'L

Figure 5. REP-PCR generated genomic ﬁngerprint. on a 1.5% agarose gel, of 15
Pantoea stewartii strains that were used in the vimlence trial. The numbered lines
with arrows below indicate the isolate of P. stewartii that is being analyzed. The
three lanes labeled with an ‘l.’ contain a 1 Kb DNA Ladder. Strains missing the 380-
bp band - Lane 6-Ohio (SW1); 7-lowa (SW 87-19); B-lowa (SW 87-6); 12-lowa (Es
9230); 13-lowa (Es 9245); 14-lllinois (SS 104). Strains with the 380-bp band- 2-
lllinois (Ps-VI-A-CP-1-1); 3-lllinois (Ps-V-A-CP-1-1); 4-lowa (SW 87-2); 5-lllinois
(SW 45); 1 O-lowa (Es 9208); 11-lowa (Es 9211); 15- Michigan (Ps-ll-B-CP-1); 16-
lllinois (Il-Ps 1); 17-ATCC-29227

37

3.5 Determination of strains based on BOX-PCR and REP-PCR. It was determined
that the 84 isolates could be separated into four different strain types, designated strain
type 1 through 4, based on their BOX-PCR and REP-PCR ﬁngerprints (Table 1). The
four strains were determined by the absence or presence of the 650-bp band in the BOX-
PCR ﬁngerprint and the 380-bp band in the REP-PCR ﬁngerprint (Figure 6). Strains
designated 1 had both of the bands. Strains designated 2 had the 650-bp BOX-PCR band,
but did not have the 380-bp REP-PCR band. Strains designated 3 had the 380—bp REP-
PCR band, but did not have the 650—bp BOX-PCR band. Strains designated 4 did not
have either band.

The REP-PCR and BOX-PCR ﬁngerprint data were combined to determine the
p-distances between the strain types 1 through 4 based on the presence of 34-36 bands.
Type 1 strains and Type 3 strains had p-distances of 0.02857, with a corresponding
similarity of 0.97143. Type 2 strains and Type 4 strains also had p-distances of 0.02857
and similarities of 0.97143. Type 1 strains and Type 3 strains had p-distances of 0.5714
from Type 2 strains and Type 4 strains, and similarities of 0.94286. The low p-distances
and high similarities between strain types reﬂect the fact that there were only two band
differences in the BOX-PCR and REP-PCR ﬁngerprint patterns.

3.6 Virulence Assessment (Leaf Symptom Analysis). Leaf symptoms of Stewart’s
bacterial wilt on leaves 1, 2 and 3 progressed over 3-6 days after initial inoculation with
15 different strains of P. stewartii (sweet corn varieties combined, Figure 7). On leaf 1,
2, and 3, the maximum disease symptom rating six days after inoculation was 0.09 (SW
87-19), 1.23 (Es 9230), and 2.69 (Es 9230), respectively. Seven out of the 15 bacterial

strains (ATCC-29227, II-B-JCP-l, Es 9211, Es 9208, SW 87-6, SW 45, SW 87-2) did not

38

-( BOX-650-bp Band)
- (REP-380-bp Band)

Type 4

- ( BOX-650—bp Band)
+ (REP-380 bp Band)

 
 
   

11:84 Type 3
+ ( BOX-650bp Band)
- (REP—380w Band)
Type 2
+ ( BOX-650-bp Band)

+ (REP-380-bp Band)

Type 1

Figure 6. The rep-PCR strain type (1, 2, 3, 4) distribution of 84
Pantoea stewartii isolates from the eastern United States (Table 1).

39

Figure 7 (A-C). Progression of Stewart’s bacterial wilt symptoms (scale 0-3 as described
in text; brieﬂy 0: no symptoms, 3: complete necrosis of leaf) on individual leaves
(Figure A-C, leaf position 1-3) from four varieties of sweet corn [J ubilee, Native Gem,
Buckeye, Ambrosia (individual points represent the average severity score of varieties
combined)] after inoculation with selected isolates of P. stewartii from Eastern USA

(listed in Table 3). + Negative control (No bacteria) ;—v-- IlEsl;

—v-- II-B-CP-l ;—-- $5104 ;—a— Es 9245 ;—o- E89230 ;—<>- Es 9211 ;
4» Es 9208 ;—A—- sw 87—6 ;—¢-- sw 87-19 ;—o-- SW1 ;—0— SW45;
—<>— sw 87-2 ;—v— V-A-CP-l-l ;---v--- VI-A-CP-l-l .

4O

A)

B)

C)

Symptoms rating

Symptoms rating

Symptoms rating

 

3.0

2.5 d

2.0 -

 

Leaf 1

 

Day 3 Day 4 Day 5 Day 6

 

0.5 ‘

0.0 —

 

Leaf 2

 

 

 

3.0

2.5 -

2.0 -

0.5 -

0.0 -‘

 

 

 

Day 3 Day 4 Day 5 Day 6

Days after inoculation

41

 

 

 

cause disease symptoms on leaf 1. The disease progression (measured by the AUDPC)
of Stewart’s bacterial wilt on sweet corn was signiﬁcantly different (p<0.05) between
leaves 1, 2 and 3 [leaf 3>2>l (Figure 8)]. When the varieties of sweet corn were analyzed
individually, the disease symptom ratings on leaf 1, leaf 2, and leaf 3 for each sweet corn
variety followed a similar trend (leaf 3>leaf 2>leaf l).

The AUDPCng. were; Jubilee (3.8), Native Gem (2.4), Buckeye (1.8), Ambrosia
(2.5). These differences were significant (p<0.05) among the four varieties of sweet corn
(Figure 9).

When all four varieties of sweet corn were combined, there were no
signiﬁcant differences (p<0.05) in AUDPC“ or AUDPC” between the 15 P. stewartii
strains, but there were signiﬁcant differences in AUDPC” (Table 5). AUDPC” of strain
Es 9230 was signiﬁcantly greater (more virulent) than strains ATCC-29227, SW 87-2,
and V-A-CP-l-l. There were also signiﬁcant differences in AUDPC”; and AUDPCm
among isolates (Table 5). Since the analysis of leaves 1, 2, and 3 combined (AUDPCLH)
and leaves 2 and 3 (AUDPCm) combined resulted in similar differences among bacterial
isolates, it was decided that because infection on leaf 1 was minimal or not infected (e.g.,
Buckeye) that leaf 1 should be excluded from subsequent analyses. AUDPCm was
therefore selected as the best indicator of Stewart’s bacterial wilt. AUDPCug type 2
strain E89230 was signiﬁcantly more virulent than type 1 strains Es 9211, Es 9208, and
SW 87-2, and type 2 strain 88104 (Table 5).

When the AUDPCLB for the 15 isolates of P. stewartii were compared within the
individual varieties of sweet corn (Jubilee, Native Gem, Buckeye, Ambrosia) there were

signiﬁcant differences (p<0.05) found between the isolates (Table 6). Within each sweet

42

 

9)

 

Fisher LSDO.05 = 0.13

.5
l

 

AUDPC Symptom Rating (Max
M

 

 

 

 

 

 

 

 

b
1 ..
9 a
o T . .
Leaf 1 Leaf 2 Leaf 3

Sweet Corn Leaf Position

Figure 8. Estimate of the area under the disease progress curve on leaves 1, 2, and 3
(AUDPCu, AUDPC”, AUDPC”) of the combined disease symptoms caused by 15
separate Pantoea stewartii strains on four different varieties of sweet com.

43

 

 

 

   

 

 

 

 

 

 

 

a? 3 Fisher LSD°_05= 0.20
:2 4' T
as
E
2’
re 3' b b
(I
E l .
2 . . c
E 2-
>~. ..
U) 2.
O
O 1 _
< 2‘:
0 1 mi ..... ,1 l l
Jubilee Native Gem Buckeye Ambrosia

Sweet Corn Variety

Figure 9. Estimate of the area under the disease progress curve of leaves 2 and 3
(AUDPCng) for the combined disease symptoms caused by 15 separate Pantoea
stewartii strains on four individual varieties of sweet corn which were inoculated near the
coleoptile and allowed to incubate for six days. Bars followed by the same letter were
not signiﬁcantly different at the p=0.05 level of probability.

Table 5. Area under the disease progress curve (AUDPC) for Stewart’s bacterial wilt
symptoms on individual leaves (AUDPCU, AUDPC”, AUDPCLz), on leaves 2 and 3
(AUDPCLB), and leaves 1, 2, and 3 (AUDPCLM) for four varieties (combined data) of

sweet corn which were rated for 4 days starting 3 days after inoculation at the 3-leaf

 

 

 

stage.t
Rep-PCR
Strain P. stewartii
Type Isolate AUDPCL." AUDPCLZ" AUDPCU' AUDPC”; AUDPCLHV
Negative Control2 0.0 a 0.0 b 0.0 C 90.0 (1 0,0 d

1 ATCC-29227 0.0 a 0.8 ab 3.7 b 2,2 c 1,5 c
1 Es 9211 0.0 a 0.8 ab 4.0 ab 2,4 c 16 c
1 Es 9208 0.0 a 1.7 a 4.9 ab 3,3 ab 2,2 ab
1 SW 45 0.0 a 1.3 a 4.3 ab 2,8 abc 1,9 abc
1 SW 87-2 0.0 a 1.0 ab 3.7 b 2,4 c 1,6 c
2 88104 0.0 a 0.8 ab 4.1 ab 2,4 c 1,6 c
2 Es 9230 0.0 a 1.7 a 5.2 a 3,5 a 2,3 a
2 SW 87-6 0.0 a 1.2 a 4.4 ab 2,8 abc 1,9 abc
2 SW 87-19 0.0 a 1.5 a 4.1 ab 2,8 abc 1,9 abc
2 SW 1 0.0 a 1.3 a 4.6 ab 2,9 abc 1,9 abc
3 [1 Es 1 0.0 a 1.5 a 4.8 ab 3,2 ab 2,1 ab
3 II-B-CP-l 0.0 a 1.1 ab 4.7 ab 2,9 abc 1,9 abc
3 V-A—CP-l-l 0.0 a 1.3 a 3.7 b 2,5 abc 1,7 abc
3 VI-A-CP—l-l 0.0 a 1.4 a 4.4 ab 2,9 abc 2,0 ab
4 ES 9245 0.1 a 1.3 a 4.6 ab 3,0 ab 2,0 ab

Degrees of freedom 511 511 511 1023 1535

Fishers LSDom 0.055 0.64 0.70 0.71 0.61

 

‘ ANOVA was performed on combined data of two trials (start dates-8 Feb and 12 Feb, 2002); means

followed by the same letter were not signiﬁcantly different at p=0.05 level of probability (T ukey multiple
comparison).

" Symptom rating analysis on Leaf 1; Max AUDPC = 9.
" Symptom rating analysis on Leaf 2; Max AUDPC = 9.
"' Symptom rating analysis on Leaf 3; Max AUDPC = 9.

" Combined symptom rating analysis of Leaf 2 and Leaf 3; Max AUDPC: 9.

’ Combined symptom rating analysis of Leaf 1, Leaf 2, and Leaf 3; Max AUDPC: 9.
' Inoculation with sterile nutrient broth.

45

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corn variety, the 15 isolates of P. stewartii were designated more virulent (MV), less
virulent (LV), or intermediately virulent (IV) based on their statistical relation (not
signiﬁcantly different at the p=0.05 level of probability) to the isolates with the highest
and lowest AUDPC”; The isolates that were consistently MV across all varieties of
sweet corn were Es 9208 (strain type 1), Es 9230 (strain type 2) and Il-Ps 1 (strain type
3). The isolates that were consistently LV across all varieties of sweet corn were ATCC-
29227 (strain type 1) and Es 9211 (strain type 1). All other isolates were not consistently
designated LV or MV on the four different varieties of sweet corn. On the most
susceptible variety of sweet corn (Jubilee), only ATCC-29227 (strain type 1), Es 9211
(strain type 1), SS 104 (strain type 2), and SW-87-6 (strain type 2) were signiﬁcantly LV
than all other isolates. On the resistant variety of sweet corn (Buckeye), members of
strain type 1; ATCC-29227, Es 9211, SW 45, SW 87-2, members of strain type 2; SS
104, SW 87-6, SW 87-19, SW 1, and members of strain type 3; VI-A-CP-l-l, V-A-CP-l-
1 were signiﬁcantly LV than the other isolates. The P. stewartii isolates ATCC-29227
(strain type 1), Es 9211 (strain type 1), SW 87-2 (strain type 1), V-A-CP-l-l (strain type
3) were LV on the two moderately susceptible sweet corn varieties, Ambrosia and Native
Gem, while Es 9208 (strain type 1), SW 45 (strain type 1), Es 9230 (strain type 2) and Il
Ps-l (strain type 3) were designated MV. The other seven strains were not consistently
MV or LV on Ambrosia and Native Gem.

3.7 Virulence Assessment (Colony Forming Units in Second Leaf Analysis) Mean
CFU/ 115 mm2 (leaf 2) were signiﬁcantly higher (p<0.05) in the Jubilee variety of sweet
corn than in Ambrosia, Native Gem, or Buckeye (Figure 10). Mean CFU/ 115 mm2

(leaf 2) in sweet corn varieties (Ambrosia, Native Gem, and Buckeye) were not

47

 

   

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Sweet Corn Variety

Figure 10. Average CFU (108) from a 115 mm2 section of leaf 2 (samples were serially
diluted to 10's) for each of the four varieties of sweet corn six days after inoculation with
15 different isolates of P. stewartii. When inoculated with isolates of P. stewartii, plants
were in a random complete block design with eight replications. Bars followed by the
same letter were not signiﬁcantly different at the p=0.05 level of probability.

48

signiﬁcantly different between strains (Figure 10). Jubilee, plants inoculated with Es
9208 had a signiﬁcantly higher mean CFU/ 115 mm2 in leaf 2 (Figure 11). No other
strains were signiﬁcantly different in terms of CFU/ 115 mm2 in leaf 2 (Figure 12, 13,
14). There were no signiﬁcantly different mean CFU / 115 mm2 in leaf 2 within Native

Gem, Buckeye, or Ambrosia.

49

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Figure 11. Average CFU (108)/ 115 mm2 of leaf tissue (samples were serially diluted to
10's) in leaf two of Jubilee sweet corn plants six days after inoculation with 15 different
isolates of P. stewartii. Brackets below (1-4) indicate the rep-PCR strain type of each
isolate. The same bracketed letters above bars were not signiﬁcantly different at p<0.05
level of probability.

50

 

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Average CFU (10°) I 115 mm"2 Native Gem leaf tissue

 

    

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Pantoea stewartii isolates

Figure 12. Average CFU (108)/ 115 mm2 of leaf tissue (samples were serially diluted to
10-3) in leaf two of Native Gem sweet corn plants six days after inoculation with 15
different isolates of P. stewartii. Brackets below (14) indicate the rep-PCR strain type
of each isolate. The same bracketed letters above bars were not signiﬁcantly different at
p<0.05 level of probability.

51

 

 

 

 

 

 

 

 

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Pantoea stewartii isolates

Figure 13. Average CFU (108)/ 115 mm2 of leaf tissue (samples were serially diluted to
10-3) in leaf two of Buckeye sweet corn plants six days after inoculation with 15 different
isolates of P. stewartii. Brackets below (1-4) indicate the rep-PCR strain type of each
isolate. The same bracketed letters above bars were not signiﬁcantly different at p<0.05
level of probability.

52

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Pantoea stewartii isolates

Figure 14. Average CFU (108) I 115 mm2 of leaf tissue (samples were serially diluted to
10's) in leaf two of Ambrosia sweet corn plants six days after inoculation with 15
different isolates of P. stewartii. Brackets below (1-4) indicate the rep-PCR strain type
of each isolate. The same bracketed letters above bars were not signiﬁcantly different at
p<0.05 level of probability.

53

CHAPTER 4

DISCUSSION

In this study, it was shown that the repetitive extragenic sequence, BOX, is
present in the genome of Pantoea and Erwinia isolates, and that the BOX-PCR protocol
can be used to differentiate these two genera. It was also demonstrated that the REP-
PCR and BOX-PCR protocols, referred to as rep-PCR collectively, could be used to
differentiate between isolates of Pantoea stewartii. Eighty-four P. stewartii isolates were
divided into four strain types (1, 2, 3, 4) based on the presence of 34—36 bands in rep-PCR
(BOX-PCR and REP-PCR) generated DNA ﬁngerprints. Type 1 strains and Type 3
strains had p-distances of 0.02857, with a corresponding similarity of 0.97143. Type 2
strains and Type 4 strains also had p-distances of 0.02857 and similarities of 0.97143.
Type 1 strains and Type 3 strains had p-distances of 0.5714 from Type 2 strains and Type
4 strains, and similarities of 0.94286. This research also explored three different
collection methods of P. stewartii isolates in and around sweet corn ﬁelds. Lastly, this
study demonstrated a rapid method for virulence assessment of the 84 P. stewartii
isolates. This method can also be used to determine sweet corn susceptibility to
Stewart’s bacterial wilt infection.

A preliminary evaluation was conducted to conﬁrm that two closely related
genera, Pantoea and Erwinia, could be differentiated from each other using the BOX-
PCR protocol. Previously, phytopathogenic species of Pseudomonas, Xanthomonas,
Clavibacter, and Ralstonia have been differentiated using the rep-PCR protocol, but

members of the genera Pantoea and Erwinia have not (Louws et al., 1994, 1998; Horita

54

and Tsuchiya, 2001). The main objective of comparing Erwinia and Pantoea isolates
was to verify that the rep-PCR protocol, speciﬁcally BOX-PCR, could be used as a tool
to differentiate between these genera. The unique genomic ﬁngerprint patterns observed
may also be a useful taxonomic tool in addition to methods described by Mergaert et al.,
(1993), (DNA-DNA hybridization and protein proﬁling) and Kwon et al., (1997), (168
rRNA gene sequences). In this study, the small number of Erwinia and Pantoea isolates
used would not allow for a complete taxonomic analysis. This preliminary trial was also
conducted to demonstrate that the rep-PCR protocol could also be used to differentiate
between two isolates (ATCC-29227 and Il-Psl) of P. stewartii.

The rep-PCR procedure was also used to differentiate 84 P. stewartii isolates into
four strain types based on their rep-PCR produced genomic ﬁngerprints using the BOX
element and repetitive extragenic palindromic (REP) primers, referred to as BOX-PCR
and REP-PCR respectively. The isolates ranged in collection dates from 1967-2001 and
collection locations (Iowa, Illinois, New York, Kentucky, Ohio, North Carolina,
Michigan). Each of the 84 isolates shared 18-19 bands (BOX-PCR) or 16-17 bands
(REP-PCR) in their rep-PCR genomic ﬁngerprints. The isolates were placed into strain
type, (1, 2, 3, 4) based on the two banding pattern differences. Type 1 strains and type 3
strains had a similarity of 97.143%. Type 2 strains and type 4 strains also had a
similarity of 97.143%. Type 1 strains and type 3 strains had a similarity of 94.286%
with type 2 strains and type 4 strains. The highly conserved relation between isolates of
P. stewartii was consistent with carbon utilization (BIOLOG) research conducted by
Wilson et al. (1999). Wilson et al. (1999) determined that a collection P. stewartii

isolates that ranged in collection dates from 1932-1994 and collection locations

55

(Delaware, Connecticut, Missouri, Iowa, Illinois, New York, Kentucky, Ohio, North
Carolina, Michigan) had a similarity of 0.93 to 1.0 based on the isolates ability to utilize
carbon sources. Five isolates that were collected during 1974 and 1975 from Ohio,
Indiana, Kentucky, and Tennessee were in both the Wilson et al. (1999) study and this
study; ATCC 29227, SW1, SW 13, SW 19, and SW 36. Wilson et al. (1999) determined
that ATCC 29227, SW 19, SW 13, and SW 36 had a similarity of 0.96 to 1.0, while SW 1
had a similarity of 0.94 to 0.96 to these four isolates. These results are in agreement with
this study where ATCC 29227, SW 13, SW 19, and SW 36 had a similarity of 1.0, and
SW 1 had a similarity of 0.94286 with the other isolates. Wilson et a1. (1999)
hypothesized that the limited variability among strains of P. stewartii could be explained
by its narrow host range, corn ﬂea beetle digestive tract and maize (Z mays), (Wilson et
al., 1999). Alternative hosts, such as grasses and weeds, have been shown to sustain P.
stewam'i populations, but it is possible that other hosts exist (Pepper, 1967; Wilson,
1999). Although an alternative host has not been identiﬁed, the proposed limited habitat
of P. stewartii favors the concept that a clonal existence is more likely than genetic strain
heterogeneity. Opportunities to introduce and sustain genomic changes caused by
recombination and mutations may be restricted because epidemics, when bacterial
populations are elevated, are infrequent. The infrequent nature of Stewart’s bacterial wilt
epidemics may also be a caused by an ‘epidemic clonal’ bacterial population. The P.
stewartii isolates in this study were collected during or within three years after the
occurrence of a Stewart’s bacterial wilt epidemic. Spratt and Maiden (1999) theorize that
prior to an epidemic, some bacteria] populations survive as avirulent or weakly virulent

pathogens, but experience some type of recombinational event that for a short period of

56

time alters the population structure causing it to be more virulent. This may be a possible
explanation for the sporadic nature of Stewart’s bacterial wilt epidemics.

The rep-PCR protocol was used by Louws et al. (1998) to detect genetic variation
in strains of Clavbacter michiganensis subsp. michiganensis. They were able to divide
this subspecies into four distinct groups (A, B, C, D) based on differences found in their
rep-PCR genomic ﬁngerprint patterns. Louws et al. (1998) also observed that 30% of the
members designated ‘A’ were nonpathogenic on tomato plants.

The variation in virulence among the strains of P. stewartii used in this research
had never been deﬁned. Therefore, a leaf symptom analysis procedure was developed
that would rapidly assess virulence of P. stewartii strains on four varieties of sweet corn
seedlings at the 3 leaf growth stage. The strains of P. stewartii for the virulence
assessment were selected to represent each rep-PCR deﬁned strain type (1, 2, 3, 4). This
was done to explore the possibility that there may be a relation between rep-PCR strain-
type and virulence on sweet corn. The four varieties of sweet corn used in this study
ranged in susceptibility from susceptible to resistant. When the data from all four
varieties of sweet corn were combined, it was determined that the area under the disease
curve for leaf 1 and leaf 2(AUDPCL1; AUDPC”), individually, were not good indicators
of virulence because there were no signiﬁcant differences observed between P. stewartii
isolates. The AUDPC“ was 0.0 for all of the isolates except Es 9245, which had an
AUDPC” of 0.1. There were signiﬁcant differences between the AUDPC of strains on
leaf 3 (AUDPCL3), but more signiﬁcant differences between isolates were found when
the AUDPC of leaf 2 and 3 or leaf 1, 2, and 3 (AUDPCLz3; AUDPCLH) were combined.

Since the AUDPC“ was 0.0 or very close to 0 and may underestimate the amount of

57

disease, the AUDPCm was determined to be the best indicator of isolate virulence.
There were signiﬁcant differences in the virulence of individual strains, (Es 9230, 1] Es 1,
Es 9245, Es 9208 were signiﬁcantly more virulent than ATCC-29227, Es 9211, SW 87-2,
SS 104), but since the more virulent isolates represent all strain types (1 , 2, 3, 4) rep-PCR
ﬁngerprints likely do not relate to virulence. The variation in virulence observed between
individual isolates may be attributed to phenotypic differences that are not genotypically
detectable by REP-PCR and BOX-PCR. When the virulence of the isolates was analyzed
on sweet corn varieties individually, there were signiﬁcant differences between the
isolates of P. stewartii. However, there was no clear trend for isolates from one rep-PCR
strain type, temporal, or geographic location to be more virulent than another on all
varieties of sweet corn. Therefore, a range of sweet corn varieties that vary in
susceptibility should be used to properly assess the virulence differentiation between
isolates of P. stewartii.

This virulence technique also showed that there were signiﬁcant differences in the
ability of the P. stewartii isolates evaluated to cause disease symptoms on the four
varieties of sweet corn (Jubilee, Native Gem, Buckeye, and Ambrosia). The
susceptibility assessment conducted by Pataky et al. (2000) rated; Jubilee as susceptible;
Native Gem as moderately susceptible; Buckeye as resistant; and Ambrosia as resistant.
The results from this study were consistent with the ratings performed by Pataky, 2000,
except in the case of Ambrosia. It was shown that the susceptibility of Ambrosia was
more closely related to Native Gem which would make Ambrosia a moderately resistant

variety rather than resistant.

58

The virulence assessment of P. stewartii isolates was rapid and therefore, may be
useful for the ﬁne screening of sweet corn varieties to determine susceptibility to
Stewart’s bacterial wilt at the seedling stage. In addition, this technique could be used to
determine the aggressiveness of P. stewartii isolates present in ﬁelds during future
epidemics. It could also enable the rapid analysis of chemical treatments, e. g.
insecticides to control corn ﬂea beetles, used to control Stewart’s bacterial wilt infection.

The assessment of colony forming units (CFU) in leaf 2 did not differentiate
between strains of P. stewartii. There were no signiﬁcant differences found among the
moderate and resistant varieties of sweet corn (Native Gem, Buckeye, Ambrosia)
analyzed and although there were signiﬁcantly higher amounts of CFU in the susceptible
variety (Jubilee) than in the moderate and resistant, only one isolate evaluated on the
variety Jubilee had signiﬁcantly higher CFU (Es 9208) in leaf 2. A relation between
virulence and the production of CFU from leaf 2 is not feasible. Although Es 9208
produced more CFU than any other isolate in Jubilee, in the combined sweet corn variety
leaf symptom virulence assessment it was not signiﬁcantly more virulent than 11 out of
the 15 isolates. Determining CFU in leaf 2 was extremely labor intensive and was not a
useful tool to ﬁnd any differences in the ability of strains to systemically infect sweet
corn with Stewart’s bacterial wilt.

The difﬁculty with which the 2001 Michigan isolate collection was assembled
raised important issues about the disease presence and importance of Stewart’s bacterial
wilt. After an entire summer of intensively searching for the casual organism of
Stewart’s bacterial wilt (P. stewartir) only three isolates were obtained and there were no

reports of Stewart’s bacterial wilt in Michigan grower’s ﬁelds. Scouting the grower’s

59

fields for symptoms of Stewart’s wilt and catching corn ﬂea beetles proved to be an
unsuccessful method of collection during a year in which the incidence of Stewart’s
bacterial wilt was minimal. Baiting with a susceptible variety of sweet corn was the only
technique that was successful, but even this method only resulted in a 1.0% rate of return.
Pepper (1967) estimated that as much as 20% of spring emerging beetles can be vectors
of P. stewartii. In epidemic years, the starting population of vectoring beetles may be
about 10%-20% (Pepper, 1967), but in years when epidemics do not occur the starting
population may be much lower, e. g. <l.0%. In such years, population density may
increase and late in the season corn ﬂea beetles may pick up inoculum that is enough to
initiate a more severe epidemic the following year. Mature plants, when infected with
Stewart’s bacterial wilt, may not show symptoms that are typical of the seedling stage of
the disease. The sporadic pattern of Stewart’s bacterial wilt outbreaks, which may be
separated by up to 30 years in Michigan, is of signiﬁcant epidemiological interest and
should continue to be monitored to determine early indicators of epidemic initiation.
Until a putative alternative host or location in which bacteria are able to persist for long
periods of time without genetic change is found that can better explain the clonal
populations of P. stewartii found in the eastern United States, prediction methods
associated with the corn ﬂea beetle’s ability to survive and carry P. stewartii should be
pursued.
Summary

Epidemic risk assessment could be based on a model that combines; a) estimation
of corn ﬂea beetle population size; b) percentage of beetles carrying P. stewartii; c) corn

ﬂea beetle prediction models that evaluate soil temperatures to better predict the number

60

of corn ﬂea beetles emerging in the spring that are carrying P. stewartii (W. Kirk, pers.
comm.). The combination of these variables may help seed companies and sweet corn
growers decide if insecticide seed treatments or foliar insecticide treatments are
warranted. The ‘epidemic clonal’ bacterial population theory by Spratt and Maiden
(1999) should also be explored. Sweet com, the digestive tract of the corn ﬂea beetle,
and other possible hosts such as weeds and grasses should be examined for the existence
of an avirulent or weakly virulent form of P. stewartii during non-epidemic growing
seasons. The rep-PCR procedure, along with the leaf symptom virulence assessment
may be used as tools to rapidly identify P. stewartii isolates. Should another series of
Stewart’s bacterial wilt epidemics occur, the virulence levels of P. stewartii isolates can
be compared to isolates that have caused epidemics in the past. Until another Stewart’s
bacterial wilt epidemic occurs or epidemics become more frequent, exploring other
genetic methods of bacterial strain differentiation may not be worthwhile because it
appears there is very little genetic diversity among P. stewartii isolates present in the

eastern United States.

61

REFERENCES

Adams, R.G. and Los, L. M. 1986. Monitoring adult corn ﬂea beetles (Coleoptera:
Chrysomelidae) in sweet corn ﬁelds with color sticky traps. Environmental
Entomology 15 (4):867-873.

Agrios, G. 1997. Plant Pathology. 4th ed. Academic Press, San Diego, CA.

Blanco, M. H., Johnson, M. G., Colbert, T. R., and Zuber, M. S. 1977. An inoculation
technique for Stewart’s wilt disease of corn. Plant Disease Reporter. 61 :413-416.

Blanco, M. H., Zuber, M. S., Wallin, J. R., Loonan, D. V., and Krause, G. F. 1979. Host-
resistance to Stewart's disease in maize. Phytopathology 69 (8):849-853.

Block, C. C., Hill, J. H., and McGee, D. C. 1998. Seed transmission of Pantoea stewartii
in ﬁeld and sweet corn. Plant Disease 82 (7):775-780.

Block, C. C., Hill, J. H., and McGee, D. C. 1999. Relationship between late-season
severity of Stewart‘s bacterial wilt and seed infection in maize. Plant Disease 83
(6):527-530.

Braun, E. J. 1990. Colonization of resistant and susceptible maize plants by Erwinia
stewartii strains differing in exopolysaccharide production. Physiological and
Molecular Plant Pathology 36 (5):363-379.

Campbell, C. L. and Madden, L. V. 1990. Introduction to Plant Disease Epidemiology.
John Wiley and Sons, New York.

Castor, L.L., Ayers, J .E., MacNab, A.A., and Krause, RA. 1975. Computerized
forecasting system for Stewart's bacterial disease of corn. Plant Disease Reporter
59 (7):533-536.

Coplin, D. L. and Majerczak, D. R. 1990. Extracellular polysaccharide genes in Erwinia
stewartii - directed mutagenesis and complementation analysis. Molecular Plant-
Microbe Interactions 3 (5):286-292.

Coplin, D. L., Frederick, R. D., and Majerczak, D. R. 1992. New pathogenicity loci in
Erwinia stewartii identiﬁed by random Tn5 mutagenesis and molecular-cloning.
Molecular Plant-Microbe Interactions 5 (3):266-268.

Coplin, D. L., Majerczak, D. R., Zhang, Y., Won-Sik, K, Jock, S., and Geider, K. 2002.

Identiﬁcation of Pantoea stewartii subsp. stewartii by PCR and strain
differentiation by PFGE. Plant Disease 86 (3):304-311.

62

dc Bruijn, FJ. 1992. Use of repetitive (repetitive extragenic palindromic and
enterobacterial repetitive intergeneric consensus) sequences and the polymerase
chain reaction to ﬁngerprint the genomes of Rhizobium meliloti isolates and other
soil bacteria. Applied and Environmental Microbiology 58:2180-2187.

Denny, T. P. 1999. Autoregulator-dependent control of extracellular polysaccharide
production in phytopathogenic bacteria. European Journal of Plant Pathology
105 (5):4l7-430.

Dillard, H. R. and Kline, W. L. 1989. An outbreak of Stewart's bacterial disease on corn
in New York State. Plant Disease 73:273.

Frutchey, CW. 1936. A Study of Stewart's disease of sweet corn caused by Phytomonas
stewarti. Degree of Doctor of Philosophy, Botany and Plant Pathology, Michigan
State College, East Lansing.

Fu, J. F., Ying, S. W., and Liu, S. T. 1997. Cloning and characterization of the ori region
of pSW1200 of Erwinia stewartii: Similarity with plasmid Pl. Plasmid 38
(3): 141-147.

Fu, J. F., Hu, J. M., Chang, Y. S., and Liu, S. T. 1998. Isolation and characterization of
plasmid pSW200 from Erwinia stewartii. Plasmid 40 (2): 100-1 12.

Fu, J. F., Chang, H. C., Chen, Y. M., Chang, Y. S., and Liu, S. T. 1996. Characterization
of the replicon of plasmid pSW500 of Erwinia stewartii. Molecular & General
Genetics 250 (6):699-704.

Gerhardt, P. 1981. Manual of Methods of General Bacteriology. American Society of
Microbiology. Washington, D. C.

Hartman, G. L. and Haywood, A. C. 1993. “Bacterial Wilt.” Proceedings of an
International Conference at Kaohsiung, Taiwan, 28-31, October, 1992. AGIAR
proceedings.

Hirano, S. S. and Upper, C. D. 1983. Ecology and epidemiology of foliar bacterial plant
pathogens. Annual Review of Phytopathology. 5:273-279.

Horita, M. and Tsuchiya, K. 2001. Genetic diversity of Japanese strains of Ralstonia
solanacearum. Phytopathology 91 (4):399-407.

Ivanoff, S. S. 1933. Stewart’s wilt disease of corn, with emphasis on the life history of
Phytomonas stewartii in relation to pathogenesis. Journal of Agricultural
Research. 47:749-770.

63

Kwon, S. G., Hee-Wan Kang, Jin-Chang Ryu, and J in-Ki Jo. 1997. Phylogentic analysis
of Erwinia species based on the 16S rRNA gene sequences. International
Journal of Systematic Bacteriology 47(4): 1061-1067.

Leigh, J. A. and Coplin, D. L. 1992. Exopolysaccharides in plant-bacterial interactions.
Annual Review of Microbiology 46:307-346.

Louws, F. J ., Schnieder, M., and de Bruijn, F. J. 1996. Assessing genetic diversity of
microbes using repetitive sequence-based PCR (rep-PCR). Pages 63-94 in:
Nuclei Acid Amplifaction Methods of Environmental Samples. Torazos G., ed.
Technomic Publishing, Co. Lancaster, PA.

Louws, F. J ., J. Rademaker, L. W., and de Bruijn, F. J. 1999. The three Ds of PCR-based
genomic analysis of Phytobacteria: Diversity, Detection, and Disease Diagnosis.
Annual Review of Phytopathology 37:81-125.

Louws, F. J., Fulbright, D. W., Stephens, C. T., and de Bruijn, F, J. 1994. Speciﬁc
genomic ﬁngerprints of phytopathogenic Xanthomonas and Pseudomonas
pathovars and strains generated with repetitive sequences and PCR. Applied and
Environmental Microbiology 60 (7)22286-2295.

Louws, F.J., Medina-Mora, C.M, Smart, CD, Opgenorth, D., Ishimaru, C.A, Hausbeck,
M.K, deBruijn, F.J., and Fulbright, D.W. 1998. rep-PCR-Mediated genomic
ﬁngerprinting: A rapid and effective method to identify Clavibacer
michiganensis. Phytopathology 88:862-868.

McGee, D. C. 1998. Maize Diseases: A reference source for seed technologists. 4th ed.
APS Press: The American Phytopathological Society. St. Paul, Minnesota.

Mergaert, J ., Verdonck, L., and Kersters, K. 1993. Transfer of Erwinia ananas
(Synonym, Erwinia uredovora) and Erwinia stewartii to the genus Pantoea
emend. as Pantoea ananas (Serrano 1928) comb. nov. and Pantoea stewartii
(Smith 1898) comb. nov., respectively, and description of Pantoea stewartii
subsp. indologenes subsp. nov. International Journal of Systematic Bacteriology
43 (l):162-173.

Munkvold, GR, McGee, DC, and Des, A. 1996. Effects of imidacloprid seed treatment
of corn on foliar feeding and Erwina stewartii transmission by the corn ﬂea
beetle. Plant Disease 80:747-749.

Pataky, J. K., du Toit, L. J ., and Freeman, N. D. 2000. Stewart's wilt reactions of an
international collection of Zea mays germ plasm inoculated with Erwinia
stewartii. Plant Disease 84 (8):901-906.

Pataky, J. K., duToit, L. J., Kunkel, T. E., and Schmitt, R. A. 1996. Severe Stewart's wilt
in central Illinois on sweet corn hybrids moderately resistant to Erwinia stewartii.
Plant Disease 80 (1): 104-104.

Pepper, Evan H. 1967. Stewart's bacterial wilt of corn. Monogr. No. 4. American
Phytopathology Society, St. Paul, MN.

Poos, F.W. 1955. Studies of certain species of Chaetocnema. Journal of Economic
Entomology 48 (5):555-563.

Poos, F.W. and Elliott, C. 1935. Bacterial wilt of corn and its insect vectors.
Phytopathology 25:32.

Purcell, A. H. and Saunders, S. 1995. Harvested grape clusters as inoculum for Pierce's
disease. Plant Disease 79 (2): 190-193.

Reddy, C. S. and Holbert , J. R. 1932 Differences in resistance to bacterial wilt in inbred
and crosses of dent corn. Phytopathology. 11:31

Schaad, N .W., Jones, J.B., and Chun, W. 2001. laboratory Guide for Identiﬁcation of
Plant Pathogenic Bacteria. 3rd ed. St Paul, Minnesota: APS Press: The American
Phytopathological Society.

Smith, J. M., Smith, N. H., O'Rourke, M., and Spratt, B. G. 1993. How clonal are
bacteria. Population Biology 90:4384-4388.

Spratt, B. G. and Maiden, M. C. 1999. Bacterial population genetics, evolution and
epidemiology. Phil. Trans. R. Soc. Lond. 354:701-710.

Stevens, NE. 1934. Stewart's disease in relation to winter temperatures. Plant Disease
Reporter 18 (12): 141-149.

Swofford, D. L. 2002. PAUP“. Phylogenetic Analysis Using Parsimony (*and Other
Methods). Version 4. Sinauer Associates, Sunderland, Massachusetts.

Thomson, S.V., Hansen, D. R., Flint, K. M., Vandenberg, J. D. 1992. Dissemination of
bacteria antagonistic to Erwinia amylovora by honey bees. Plant Disease 76
(10): 1052-1056.

Versalovic, J., Schneider, M., de Bruijn, F. J ., and Lupski, J .R. 1994. Genomic
ﬁngerprinting of bacteria using repetitive sequence-based polymerase chain
reaction. Methods in Molecular and Cellular Biology 5:25-40.

Wilson, W. J. and H. R. Dillard. 1999. Assessment of phenotypic variability in Erwinia
stewartii based on metabolic proﬁles. Plant Disease 83 (2):]14-118.

65

APPENDICES

66

APPENDIX A

Optimization of BOX primer for rep-PCR (Example)

67

     

“E‘W

' ‘UU LTUUT

11 ‘15 L

 

Figure 15. Optimization of Rep-PCR using the BOX primer generated genomic ﬁngerprint
of Pantoea stewartii strains (ATCC-29227 and Il-Ps 1). The numbered brackets below
indicate the strain of P. stewartii and levels of H20 that is being analyzed. Each strain and
H10 alteration is repeated in adjacent lanes and represented by the same number. The three
lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2—Il-Ps l ( 12.25 pl of H20); 3—
ATCC-29227 (12.25 pl of H10); 4-Il-Ps 1 (12.30 pl of H20); 5 ATCC-29227 (12.30 pl of
H20); 6-Il-Ps 1 ( 12.35 pl of H20); 7-ATCC-29227 ( 12.35 pl of H20); 8-Il-Ps 1 (12.40 pl of
H20); 9-ATCC-29227 (12.40 pl of H20); 10-Il-Ps l (12.45 pl of H20); 11-ATCC-29227
(12.45 pl of H20); 12-Il-Ps l (12.50 pl of H20); l3-ATCC-29227 (12.50 pl of H20); 14-11-
Ps 1 (12.55 pl of H20); lS-ATCC-29227 (12.55 pl of H20).

68

APPENDIX B

Optimization of REP primer for rep-PCR (Example)

69

1636b

1018b

517 b

 

Figure 16. Optimization of Rep-PCR using the REP primer generated genomic ﬁngerprint
of Pantoea stewartii strains (ATCC—29227 and Il-Ps l). The numbered brackets below
indicate the strain of P. stewartii and levels of H20 that are being analyzed. Each strain
and H20 alteration is repeated in adjacent lanes and represented by the same number. The
three lanes labeled with an ‘L‘ contain a 1 Kb DNA Ladder. Lane 2-ATCC-29227 (12.45
pl of H20); 3-Il-Ps 1 (12.45 pl of H20); 4 ATCC-29227 (12.50 pl of H20); 5-Il-Ps 1 (12.50
pl of H20); 6-ATCC-29227 (12.55 pl of H20); 7-Il-Ps 1 (12.55 pl of H20); 8-ATCC-
29227 (12.60 pl of H20); 9-Il-Ps l (12.60 pl of H20); 10-ATCC-29227 (12.65 pl of H20);
ll-Il-Ps 1 (12.65 pl of H20).

70

APPENDIX C

Identiﬁcation of CFU using Rep-PCR and the BOX primer

71

 

Figure 17. rep-PCR using the BOX primer generated genomic fingerprint. on a 1.5%
agarose gel, of 15 Pantoea stewartii strains that were randomly selected as checks to
conﬁrm that the colonies counted in the virulence trial were P. stewartii. The
numbered lines with arrows below indicate the isolate of P. stewartii that is being
analyzed. The three lanes labeled with an ‘L' contain a 1 Kb DNA Ladder. Strains
missing the 650-bp band - Lane 2-lllinois (Ps-Vl-A-CP-1-1); 3-lllinois (Ps-V-A-CP-1-
1); 13—Iowa (Es 9245): 15- Michigan (ll-B-CP-1); 16- Illinois (ll-Ps 1). Strains with the
650-bp band- Lane 4-Iowa (SW 87-2); 5-lllinois (SW 45); 6-Ohio (SW 1); 7-lowa (SW
87-19); 8-lowa (SW 87-6); 10-Iowa (Es 9208); 11-lowa (Es 921 1); 12-lowa ( Es 9230);
14-lllinois (SS 104); 17-ATCC-29227

72

APPENDIX D

Rep-PCR using the BOX primer gels with 84 isolates of P. stewartii

73

1636 bp

1018 bp

517 bp

 

Figure 18. Rep-PCR using the BOX primer generated genomic ﬁngerprint,
on a 1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets
below indicate the strain of Pantoea stewartii that was analyzed. Each
strain is repeated in adjacent lanes and represented by the same number.
The three lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2
and 15 contain the positive controls- ATCC-29227 and Illinois-control (Il-
Ps-l). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-control
(Il-Ps-l), respectively. 3-Illinois (VIII-A-CP-3—l); 4-Illinois (VI-A-CP-Z-
1); S-Illinois (V -A-CP-4-2); 6-Illinois (VIII-A-CP-3-2); 7-Michigan (II-B-
CP-l); lO-Illinois (V -A-CP-l-2); 11-Illinois (VI-A-CP-2-3); 12-Illinois
(VI-A CP-1-3); 13-Illinois (VI-A-CP-1-2); l4-Illinois (VI-A-CP-l-l).

74

1 ' anew». 4M-{w4rx-

1.
3
.
§
.
‘:
E
I
.s
r
I

 

Figure 19. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5%
agarose gel. of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L’ contain a 1 Kb
DNA Ladder. Lanes 2 and 15 contain the positive controls- Illinois-control (Il-Ps-l) and
ATCC-29227. Lane 8 and 9 also contain strain Illinois-control (Il- Ps-l) and ATCC-
29227, respectively. 3-Iowa (SW 87-1); 4-Iowa (SW 87-2); S-Ohio (SW1); 6-Illinois (V-
A-CP-l-l); 7-Illinois (V-A-CP-3-2); lO-Illinois (V-A-CP-B-l); ll-Illinois (V-A-CP-4-1);
12-Iowa (SW 87-3); l3-Iowa (SW 87-4); l4-Illinois (VI-A-CP-4-l).

75

..A
U'
‘U

..a hug-.... an ‘1:

1018 bp->

517 bp—f

 

. «m. r. rm. ....w.u.‘n.w

Figure 20. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L’ contain a 1
Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and
Illinois-control (Il-Ps-l). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-
control (Il-Ps-l), respectively. 3-Iowa (SW 87-23); 4-Iowa (SW-87-22); 5-Iowa (SW-
87-21); 6-Iowa (SW-87-20); 7-Iowa (SW-8749); lO-Iowa (SW-8748); ll-Iowa (SW-
87-9); 12-Iowa (SW-87-7); l3-Iowa (SW-87-25); 14-Iowa(87-24).

76

  
  
    

1636bp—D‘

       

—' 1

. ' .. , I: ‘m
3 4 56 78L9101112131415L
Figure 21. Rep—PCR using the BOX primer generated genomic ﬁngerprint, on
a 1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets
below indicate the strain of Pantoea stewartii that was analyzed. Each strain is
repeated in adjacent lanes and represented by the same number. The three
lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain
the positive controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9
also contain strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively.
3-Iowa (SW-87-l7); 4-Iowa (SW-87-16); 5-Iowa (SW-8745); 6-Iowa (SW-
87-13); 7-Iowa (SW 87-12); lO—Iowa (SW 87-11); ll-Iowa (SW 87-10); 12-
Iowa (SW-87-8); l3-Iowa (SW-87-5); l4-Iowa (SW-87-6).

77

 

4 5 6 78L9101112131415L

Figure 22. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled
with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive
controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also contain
strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es
9225); 4-Iowa (Es 9230): S-Iowa (Es 9243); 6-Iowa (Es 9244); 7-Iowa (Es
9245); lO—Iowa (Es 9222); ll-Iowa (Es 9221); 12-Iowa (Es 9203); l3-Iowa (Es
9204); 14-Iowa (Es 9205).

78

1636 bp—> " '

   
 
  

1018 bp —>

517bp—>

 
 

r \
l ‘1

h
7

l

 

I r‘ ,‘!.'. 1‘1 ;; r‘ i“ .,
L h i n i
2 3 4 5 6

8L9101112131415L

Figure 23. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated in
adjacent lanes and represented by the same number. The three lanes labeled with
an ‘L' contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls-
Illinois-control (Il- Ps-l) and ATCC-29227. Lane 8 and 9 also contain strain
Illinois-control (Il- Ps-l) and ATCC-29227, respectively. 3-Illinois (SS 104) . 4-
North Carolina (SW 39); 5-Iowa (Es 9250); 6-Iowa (Es 9256); 7-Tennessee (SW
36); lO-Iowa (Es 9253); ll-Iowa (Es 9245); 12-Indiana (SW 14); l3-Iowa (Es
9247); l4-Illinois (SW 45).

79

ill.

1636 bp —-

101a bp—>...

 

Figure 24. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on
a 1.5% agarose gel. of Pantoea stewartii strains. The numbered brackets
below indicate the strain of Pantoea stewartii that was analyzed. Each strain is
repeated in adjacent lanes and represented by the same number. The three
lanes labeled with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain
the positive controls - Illinois-control (Il-Ps-l) and ATCC-29227. Lane 8 and
9 also contain strain Illinois-control (II-Ps-l) and ATCC-29227, respectively.
3-Indiana (SW 13); 4-0hio (SW 2); 5-Ohio (Rif EsA); 6-Iowa (Es 9257); 7-
Ohio (SW 1); lO-Iowa (Es 9254); ll-Kentucky (SW 19); 12-Iowa (Es 9248);
13—Iowa (Es 9249); 14-Iowa (9246).

80

 

Figure 25. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled
with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive
controls- ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also contain
strain ATCC-29227 and Illinois-control (Il-Ps-l), respectively. 3—Iowa (Es 9203);
4-Iowa (Es 9204); 5 Iowa (Es 9205); 6-Iowa (Es 9206); 7-Iowa (Es 9207); 10-
Iowa (Es 9208); ll-Iowa (Es 9209); 12-Iowa (Es 9211); 13-Iowa (Es 9210); 14-
Iowa (Es 9201).

81

1636 hp

1018 bp

 

Figure 26. Rep-PCR using the BOX primer generated genomic
ﬁngerprint, on a 1.5% agarose gel, of Pantoea stewartii strains. The
numbered brackets below indicate the strain of Pantoea stewartii that was
analyzed. Each strain is repeated in adjacent lanes and represented by the
same number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA
Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and
Illinois-control (Il-Ps-l). Lane 8 and 9 also contain strain ATCC-29227
and Illinois-control (Il-Ps-l), respectively. 3-Iowa (Es 9223); 4-Iowa (Es
9222); S-Iowa (Es 9221); 6-Iowa (Es 9213); 7-Iowa (Es 9212); lO—Iowa
(Es 9264); ll-Iowa (Es 9244); 12-Iowa (Es 9243); 13-Iowa (Es 9246); 14-
Iowa (Es 9230).

82

1636 bp

1018bp

517 bp

 

IE;._ IEis Ea

.‘u

4 5 6 718L910 '11"12 1514 1‘ L

Figure 27. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L’ contain a 1
Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- Illinois-control (Il-Ps—l)
and ATCC-29227. Lane 8 and 9 also contain strain Illinois-control (Il-Ps-l) and
ATCC-29227, respectively. 3-Illinois (SW 45); 4-Kentucky (SW 19); 5-Ohio (GAL
E8); 6-0hio (DC 130); 7-Ohio (DC 160); lO-Ohio (MU 51); ll-Indiana (SW 13); 12-
Indiana (SW 14); 13-Ohio (DC 283); l4-Michigan (II-B-CP-7).

83

 

Figure 28. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated in
adjacent lanes and represented by the same number. The three lanes labeled with an
‘L' contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls-
ATCC-29227 and Illinois-control (Il-Ps-l). Lane 8 and 9 also contain strain ATCC-
29227 and Illinois-control (Il-Ps-l), respectively. 3—Iowa (Es 9249); 4-Iowa (Es
9247); S-lowa (Es 9248); 6-Iowa (Es 9250): 7-Iowa (Es 9253); lO-Iowa (Es 9256);
ll-Ohio (Rif Es 9A); 12-Illinois (SS 104); l3-Iowa (Es 9264); 14-Iowa (Es 9257).

1636 bp

1018bp

517 bp

 

Figure 29. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L' contain a 1
Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and
Illinois-control (Il-Ps-l). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-
control (Il-Ps-l), respectively. 3-Iowa (Es 9213); 4-Iowa (Es 9212); 5-Iowa (E5 9211);
6-Iowa (Es 9210); 7-lowa (Es 9206); lO-Iowa (Es 9207); ll-Iowa (Es 9208); l2-Iowa
(Es 9209); l3-Iowa (SW 87-26); 14-lowa (Es 9201).

85

 

   
    

1636 bp

1018bp

517 rap—ml. 1'

010111213 1415 L17 1! 1920 2122232425 26 272829 L

Figure 30. Rep-PCR using the BOX printer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbers below the arrows
indicate the strain of Pantoea stewartii that was analyzed. The three lanes labeled
with an ‘L' contain a 1 Kb DNA Ladder. Lanes 2 and 3 contain the positive
controls- Illinois-control (Il-Ps-l) and ATCC-29227, respectively. Lane 17
contains the ATCC-29227 control. Lane 28 and 29 also contain strain Illinois—
control (Il-Ps-l) and ATCC-29227. respectively. 4-Iowa (SW 87-11); 5-Iowa (SW
87-12); 6-Iowa (SW 87-13); 7 Iowa (SW 87-15); 8 Iowa (SW 87-1); 9-Ohio (DC
130); lO-Ohio (DC 160); ll-North Carolina (SW 39); 12—Illinois (SW 45); 13-
Tennessee (SW 36); l4-Kentucky (SW 19); lS-Iowa (SW 87-16); lS-Iowa (SW
87-17); l9-Iowa (SW 87-18); 20—Iowa (SW 87-19); 21-Iowa (SW 87-20); 22-Iowa
(SW 87-21); 23-Iowa (SW 87-26); 24-Iowa (SW 87-25); 25-Iowa (SW 87-24); 26-
Iowa (SW 87-23); 27-Iowa (SW 87-22).

86

'vrm. u s "’3' d... ‘uox—nau— -->

 

L 2 345678 91011L131415161718192021 L

Figure 31. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbers below the arrows indicate the
strain of Pantoea stewartii that was analyzed. The three lanes labeled with an ‘L’
contain a 1 Kb DNA Ladder. Lane 2 contains the positive control- ATCC-29227. Lane
13 contains the Illinois control (11 Ps-l). Lane 21 contains ATCC-29227. 3-Ohio (DC
283); 4-Ohio (SW 1); 5-0hio (MU 51); 6-Ohio (GAL E8); 7-Indiana (SW 14); 8-
Indiana (SW 13); 9-Ohio (SW 2); lO-Iowa (SW 87-2); “-10er (SW 87-3); l4-lowa
(SW 87-4); 15-Iowa (SW 87-5); 115-Iowa (SW 87-6); 17-Iowa (SW 87-7); 18-Iowa (SW
87-8); l9-Iowa (SW 87-9); 20-Iowa (SW 87-10).

87

517bp—9

 

I I I
L 2 3 4

Figure 32. Rep-PCR using the BOX primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an 'L' contain a 1
Kb DNA Ladder. Lanes 2 and 10 contain the positive controls— Illinois-control (Il-Ps-l)
and ATCC-29227. Lane 5 and 6 also contain strain Illinois-control (Il-Ps-l) and
ATCC-29227, respectively. 3-Illinois (V-A-CP—4-1);4-Illinois (VIII-A-CP-3-2); 7-
lllinois (V III-A-CP-3- l ); 8-Illinois (Vl-A-CP-2-1); 9-Illinois (Vl-A—CP-4- l ).

88

  

1636 bp—V {j

517 bp

    

I I! I I:
4 5 6 7 8 L 9 10 11 12 13 14 15 L

Figure 33. Rep-PCR using the BOX primer generated genomic fingerprint, on a 1.5%
agarose gel. of Pantoea stewartii strains. The numbered brackets below indicate the strain
of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes and
represented by the same number. The three lanes labeled with an ‘L’ contain a 1 Kb DNA
Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and Illinois-control
(Il-Ps-l). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-control (Il-Ps-l),
respectively. 3-Illinois (V I-A-CP—l-2); 4-Illinois (V I-A-CP-l-3); S-Illinois (V-A-CP4-2);
6-Michigan (II-B-CP-S); 7-Illinois (VI-A-CP-l-3); lO-Illinois (V-A-CP-3-2); ll-Illinois
(V-A-CP-l-l); lZ-lllinois (V-A-CP-3-l); l3-Illinois (V -A-CP-l-2); l4-Illinois (VI-A-CP-
l-l).

89

APPENDIX E

Rep-PCR using the REP primer gels with 84 isolates of P. stewartii

90

1636bp

1018bp

517 bp

 

Figure 34. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled with
an ‘L' contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls-
ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain
ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Illinois (VIII-A-CP-S-
1); 4-lllinois (VI-A-CP-Z-l); S-Illinois (V-A-CP-4-2): 6-Illinois (VIII-A-CP-3-2);
7-Michigan (II-B-CP—l); lO—Illinois (V-A-CP-l-2); ll-Illinois (VI-A-CP-2-3); 12-
Illinois (VI-A CP~l-3); 13—Illinois (VI-A-CP-l-Z); l4-Illinois (VI-A-CP-l-l).

91

 

Figure 35. Rep-PCR using the REP primer generated genomic ﬁngerprint. on a
l.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated in
adjacent lanes and represented by the same number. The three lanes labeled with an
‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls-
ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-
29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (SW 87-l); 4-Iowa (SW-
87-2); S-Ohio (SW 1); 6—Illinois (V -A-CP— l-l); 7-Illinois (V -A-CP-3-2); lO—Illinois
(V-A-CP-3-l); ll-Illinois (V-A-CP-4-1); 12-Iowa (SW-87-3); l3-Iowa (SW-874);
l4-Illinois (VI-A-CP-4-1).

92

 

Figure 36. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel. of Pantoea stewanii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L' contain a 1
Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and
Illinois-control (II-Ps 1). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-
control (Il-Ps 1), respectively. 3-Iowa (SW 87-23); 4-Iowa (SW-87-22); S-Iowa (SW-
87-15); 6-Iowa (SW 87-13); 7-Iowa (SW-87-12); lO-Iowa (SW-87-11); ll-Iowa (SW
87-10); 12-Iowa (SW-87-8); 13-Iowa (SW-87-5); l4-Iowa (SW 87-6).

93

 

Figure 37. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L’ contain a 1
Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and
Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-
control (Il-Ps 1), respectively. 3-lowa (SW 87-17); 4-Iowa (SW-87-16); 5~Iowa (SW-
87-21); 6-Iowa (SW 87-20); 7-Iowa (SW-87-l9); lO-Iowa (SW-87-l8); ll-Iowa (SW
87-9); 12-Iowa (SW-87-7); l3-lowa (SW-87-24); l4-lowa (SW 87-25).

94

1636bp

     

i . eras '10. 31:1 I 1213 14 15 h

r 23%

Figure 38. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel. of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewam'r' that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L' contain a 1 Kb
DNA Ladder. Lanes 2 and l5 contain the positive controls- ATCC-29227 and Illinois-
control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-control (Il-Ps
1), respectively. 3-Iowa (Es 9257); 4-Iowa (Es 9256); S-Iowa (Es 9253); 6-Iowa (Es
9250); 7-Iowa (Es 9249); lO-Iowa (Es 9248); ll-Iowa (Es 9247); lZ—Kentucky (SW 19);
l3-Indiana (SW 2); l4-Indiana (SW 13).

95

 

Figure 39. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewanii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L' contain a 1
Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and
Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-
control (Il-Ps 1). respectively. 3-Iowa (Es 9213); 4-Iowa (Es 9212); 5-Iowa (Es 9211);
6-Iowa (Es 9210); 7-Iowa (Es 9206); lO-Iowa (Es 9207); ll-Iowa (Es 9208); l2-Iowa
(Es 9209); l3-Iowa (SW 87-26); l4-Iowa (Es 920l).

96

1018 bp —>; ..

t

517bp J.-

 

”C:
“‘3
AE: 5' '
“C: '
“E:
“C:
as.

Figure 40. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled
with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive
controls- ATCC-29227 and IllinoiSocontrol (Il-Ps 1). Lane 8 and 9 also contain
strain ATCC-29227 and Illinois-control (Il-Ps 1). respectively. 3-Iowa (Es 9249);
4-lowa (Es 9247); 5-Iowa (Es 9248); 6-Iowa (Es 9250); 7-Iowa (Es 9253); lO—Iowa
(Es 9256); ll-Ohio (Rif Es 9A); lZ-Illinois (SS 104); l3-Iowa (Es 9264); l4-Iowa
(Es 9257).

97

 

Figure 41. RepoPCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent
lanes and represented by the same number. The three lanes labeled with an ‘L‘
contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-
29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227
and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9243); 4-Iowa (Es 9240); 5-
Iowa (E3 9225); 6-Iowa (Es 9230); 7-Iowa (Es 9222); lO-Iowa (Es 9221); ll-Iowa
(Es 9213); lZ-lowa (Es 9244); lS-Iowa (Es 9245); 14-Iowa (Es 9246).

98

517bp -> -

 

Figure 42. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled
with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive
controls- ATCC-29227 and Illinois—control (Il-Ps 1). Lane 8 and 9 also contain
strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Ohio (DC 283);
4-Ohio (DC 160); 5-Ohio (DC 130); 6-Iowa (SW 87-8); 7-Iowa (SW-87-7); 10-
lowa (SW-876); ll-Indiana (SW 14); l2-Tennessee (SW 36); 115-North Carolina
(SW-39); l4-Illinois (SW 45).

99

1636bp—> ...

101abp—>

517 bp —> 1'

 

 

Frgure 43. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent
lanes and represented by the same number. The three lanes labeled with an ‘L'
contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-
29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227
and Illinois-control (Il-Ps 1), respectively. 3-Michigan (II-B-CP-l); 4-Iowa (SW-87-
4); 5-Iowa (SW-876); 640% (SW 87-18); 7-Iowa (SW-87-l7); lO—Iowa (SW-87-
3); ll-Iowa (SW 87-2); lZ-Michigan (II-B-CP-7); 13-Iowa (SW-87-l); 14-Michigan
(II-B-CP—S).

100

       
   
    

JM‘K“Wxauﬁrirlu-W"~W‘

.1er mad... ,

’PWI

   

W’“” '1'? “awn—1.1m Mu. .

it.

v new—u“ mu...

1636 bp —>
““Rﬁiazeéa <
‘Q‘F‘E‘V'r' *a—ﬁ'

”wha- *1

15L

Figure 44. Rep—PCR using the REP primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewanir' strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled
with an ‘L’ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive
controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain
strain ATCC-29227 and Illinois-control (Il-Ps 1), respectively. 3-Iowa (Es 9246);
4-Iowa (Es 9212); 5-Iowa (Es 9210); 6-Iowa (Es 9208); 7-Iowa (Es 9207); 10-
Iowa (Es 9206); ll-Ohio (SS 104); 12-Iowa (Es 9264); 13-Ohio (DC 283); 14-
Ohio (MU 51).

101

1636bp—>

1018bp->

517bp —>

 

 

L234567L8""10111213L
Figure 45. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel. of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L' contain a 1 Kb
DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and Illinois-
control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-control (Il-
Ps 1), respectively. 3-lllinois (VI-A—CP-l-l); 4-(SW 45); 5- (SW 39); 6—Illinois (V -A-
CP-3—2); 9-Illinois (VIII-A-CP-3-2); 10-Illinois (V-A-CP-4-2); ll-Illinois (VI-A CP—2—
l); 12-Illinois (VI-A-CP-4-1).

102

     

517bp —>'

      

m ‘ ,

L 2 3 ' 4
Figure 46. Rep-PCR using the REP primer generated genomic ﬁngerprint,
on a 1.5% agarose gel. of Pantoea stewartii strains. The numbered brackets
below indicate the strain of Pantoea stewartii that was analyzed. Each strain
is repeated in adjacent lanes and represented by the same number. The three
lanes labeled with an ‘L' contain a 1 Kb DNA Ladder. Lanes 2 and 15
contain the positive controls- ATCC-29227 and Illinois-control (Il-Ps 1).
Lane 8 and 9 also contain strain ATCC-29227 and Illinois-control (Il-Ps 1),
respectively. 3-Illinois (VI-A-CP-2-3); 4-Illinois (V-A-CP-l-Z); 5- Illinois
(V-A-CP-3-l); 6-Illinois (V-A-CP—l-l); 9-Illinois (V-A-CP-4-1); lO-Illinois
(VI-A-CP-l-3); 11-Illinois (VI-A CP-1-2); 12-lllinois (V IH-A-CP-3-l).

5 “3'6 7’1 "’8 10- 111912 13 "L

103

1636bp—>

1018bp—>

517bp —>

 

 

Figure 47. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a
1.5% agarose gel, of Pantoea stewartii strains. The numbered brackets below
indicate the strain of Pantoea stewartii that was analyzed. Each strain is repeated
in adjacent lanes and represented by the same number. The three lanes labeled
with an ‘L‘ contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive
controls- ATCC-29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain
strain ATCC-29227 and Illinois~control (Il-Ps 1), respectively. 3-Ohio (GAL E8);
4-0hio (MU 51); S-Illinois (SW 45); 6-North Carolina (SW 39); 7—Tennessee
(SW-36); lO-Indiana (SW 14); ll-Ohio (DC 130); 12-Ohio (DC 160); l3-Ohio
(DC 283); 14-Ohio (SW 1).

104

517bp —>

.14 15 51*,

 

 

Figure 48. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent
lanes and represented by the same number. The three lanes labeled with an ‘L'
contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-
29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227
and Illinois~control (Il-Ps 1), respectively. 3-Ohio (GAL E8); 4-Ohio (MU 51); 5-
Illinois (SW 45); 6- North Carolina (SW 39); 7-Tennessee (SW-36); lO-Indiana (SW
14); ll-Ohio (DC 130); 12-0hio (DC 160); l3-Ohio (DC 283); l4-Ohio (SW 1).

105

 

Figure 49. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel. of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent lanes
and represented by the same number. The three lanes labeled with an ‘L' contain a 1 Kb
DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-29227 and Illinois-
control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227 and Illinois-control (Il-
Ps 1), respectively. 3-Iowa (87-13); 4-Iowa (87-15); 5-Iowa (87-16); 6-lowa (87-20); 9-
Iowa (87-19); lO-Iowa (87-22); ll-Iowa (87-21); 12-Iowa (87-6).

106

1636 bp->

    

101a bp—>

a

7L8-9 10111213 L

Figure 50. Rep-PCR using the REP primer generated genomic ﬁngerprint, on a 1.5%
agarose gel, of Pantoea stewartii strains. The numbered brackets below indicate the
strain of Pantoea stewartii that was analyzed. Each strain is repeated in adjacent
lanes and represented by the same number. The three lanes labeled with an ‘L'
contain a 1 Kb DNA Ladder. Lanes 2 and 15 contain the positive controls- ATCC-
29227 and Illinois-control (Il-Ps 1). Lane 8 and 9 also contain strain ATCC-29227
and Illinois-control (Il-Ps 1), respectively. 3-Iowa (87—7); 4-Iowa (87-8); 5-Iowa
(87—18); 6-Iowa (87-17); 9-Iowa (87-9); 10-Iowa (87-10); ll-Iowa (87-11); 12-Iowa
(87-12).

    

107

IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

lllllljlljlﬂﬂjllmﬂljljljl[lilﬂjjll