The causal agent of fire blight of pear and apple. Alyssa Carey
Dr. Joyce Loper Dr. Virginia Stockwell Erwinia amylovora 1 to 2 m
long rod-shaped bacterial cells
Slide 2
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
Slide 3
Spring Cankers activate, Erwinia amylovora spread by bees and
rain and grows on flower tissues Early Summer Infection of blossoms
Shoot and fruit infection Summer and Fall Pathogen kills branches
and forms cankers Winter Pathogen overwinters in cankers Disease
Cycle of Fire BlightX
Slide 4
Research Opportunities 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
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Slide 5
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
Slide 6
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
Slide 7
Hypothesis Erwinia amylovora isolates from the Pacific
Northwest contain extrachromosomal DNA in addition to pEA29
Slide 8
Goals Isolate and characterize plasmids of Erwinia amylovora
from the Pacific Northwest Determine if there is a correlation of
plasmid content and orchard location
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1162548310
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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
Slide 10
RFLP (Restriction Fragment Length Polymorphism) Analysis of
Plasmids Using restriction enzyme digests to identify unique and
characteristic patterns EcoRI BamHI
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pEA29 Use restriction enzymes to examine plasmid preparations
28,185 bpsuse sequence to predict fragment sizes If extra bands
Mutations Other incorporated DNA in pEA29 Other extrachromosomal
DNA (plasmid) in isolate BamHIEcoRI
Slide 12
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
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Slide 13
Locations of orchards with isolates of E. amylovora lacking
pEA29 Yakima Valley, WA Sampled orchard Orchard with E. amylovora
without pEA29
Slide 14
Additional Plasmids? About half of 205 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 EcoRI digest
Slide 15
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.
Slide 16
Locations of orchards with isolates that were PCR positive for
pEU30 Yakima Valley, WA Sampled orchard Orchard with E. amylovora
with pEU30
Slide 17
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
Slide 18
Summary 205 isolates of E. amylovora Over half had altered
plasmid profiles 6 lacked pEA29 27 were positive for pEU30
Slide 19
Acknowledgments Mentor Virginia Stockwell Lab Members Joyce
Loper Marcella Henkels Brenda Shaffer HHMI Kevin Ahern Gayle McGhee
Michigan State University Larry Pusey USDA-ARS, Wenatchee, WA
Krishna Mohan University of Idaho, Parma Kathleen McNamara Bear
Creek Orchards, Medford, OR
