Erwinia amylovora
1 to 2 μm long rod-shaped
bacterial cells
The causal agent of fire blight of pear and apple.
Alyssa Carey
Dr. Joyce Loper
Dr. Virginia Stockwell
Fire Blight


Most severe bacterial
disease of pear and
apple
First discovered in
Hudson Valley – 1780.
 Spread across US, and to
Europe, the Middle East and
New Zealand

Economically devastating
in all areas.
 Pacific Northwest
○
1988 - $89 million (Central WA)
○ 1998 - $68 million (Hood River Valley)
○ 2002 – Worst epidemic in 100 years
Medford, OR
Disease Cycle of Fire Blight
Spring
Cankers activate,
Erwinia amylovora spread by
bees and rain and grows on
flower tissues
X
Winter
Early Summer
Pathogen overwinters
in cankers
Infection of blossoms
Shoot and fruit infection
Summer and Fall
Pathogen kills
branches and forms
cankers
Research Opportunities



http://www.sorengallery.com/images/ra20gp2040x48.jpg
Periodic fire blight epidemics
continue to damage orchards
causing great losses
There are few options for
disease control
Erwinia amylovora long
considered homogeneous but
recent findings indicate that
the pathogen may be more
diverse
Summer Research



Characterize isolates of
Erwinia amylovora from
orchards in the Pacific
Northwest
Determine if the plasmid
pEA29 is ubiquitous in
Northwest isolates
Determine if other
extrachromosomal DNA is
present in Northwest
isolates of fire blight
pathogen
http://www.plantpath.cornell.edu/Labs/Beer/images/photos/Eamyl
ovora.gif
pEA29



Considered near
ubiquitous in Erwinia
amylovora.
Associated with
virulence/fitness, but not
essential
Target for some PCR
methods for
identification/detection of
E. amylovora
Hypothesis

Erwinia amylovora isolates from the Pacific
Northwest contain extrachromosomal DNA in
addition to pEA29
Goals


Isolate and
characterize plasmids
of Erwinia amylovora
from the Pacific
Northwest
Determine if there is a
correlation of plasmid
content and orchard
location
http://farm1.static.flickr.com/122/280920864_5065ccb7e3.jpg?v=
1162548310
Procedure

Obtain samples from
orchards
 Dilution series
 Plate onto agar medium
for growth

Confirm identity as
Erwinia amylovora
 PCR reactions
 AgriStrip test
 Sequencing of 16S rRNA

Isolate plasmid DNA
using alkaline lysis
method
 PCR reactions for pEA29
 Restriction digest or
RFLP analysis
RFLP (Restriction Fragment Length
Polymorphism) Analysis of Plasmids

Using restriction enzyme
digests to identify unique
and characteristic patterns
 EcoRI
 BamHI
http://cleaver.sourceforge.net/index_files/CleaverIcon3Bitmap.png
BamHI EcoRI
pEA29

Use restriction enzymes to examine
plasmid preparations
 28,185 bps—use sequence to predict fragment sizes
 If extra bands
○ Mutations
○ Other incorporated DNA in pEA29
○ Other extrachromosomal DNA (plasmid) in isolate
Isolates without pEA29

pEA29 considered
ubiquitous in United
States
 A few isolates lacking
pEA29 recorded in
Iran, Egypt and EU

From our collection
 Six isolates of 205
examined (3%) lack
this plasmid
http://farm3.static.flickr.com/2324/2065568371_c2bfc1c0b7.jpg?v=0
Yakima Valley, WA

Locations of orchards
with isolates of E.
amylovora lacking
pEA29
Sampled orchard
Orchard with E. amylovora
without pEA29
Additional Plasmids?
About half of 205 M
isolates have more
bands in addition to
those from pEA29 in
RFLP
 Unique banding patterns
indicate that additional
plasmid(s) exist within
PNW isolates of Erwinia
amylovora
M
pEA29

M
EcoRI digest
pEU30




Another plasmid in Utah
strain of E. amylovora
found by Foster et al.
pEU30 was sequenced
and primers for detection
developed by Foster
27 (13%) of our strains
were positive with primers
designed to this plasmid
Isolates with pEU30 also
had pEA29
Foster, McGhee, Jones, and Sundin. 2004.
Appl. Environ. Microbiol. 70:7539-7544.
Yakima Valley, WA

Locations of
orchards with
isolates that were
PCR positive for
pEU30
Sampled orchard
Orchard with E. amylovora with
pEU30
Current Research Activities



Sequencing an
uncharacterized
plasmid(s) from my
collection
Four isolates with unique
RFLP patterns were
selected
Isolates were cured of
pEA29 using eviction
mutagenesis
Summary
205 isolates of E. amylovora
Over half had altered plasmid profiles
– 6 lacked pEA29
– 27 were positive for pEU30
Acknowledgments

Mentor


Virginia Stockwell
Lab Members
 Joyce Loper
 Marcella Henkels
 Brenda Shaffer

HHMI


Kevin Ahern
Gayle McGhee
 Michigan State University

Larry Pusey


Krishna Mohan


USDA-ARS, Wenatchee, WA
University of Idaho, Parma
Kathleen McNamara

Bear Creek Orchards, Medford, OR