The causal agent of fire blight of pear and apple. Alyssa Carey Dr. Joyce Loper Dr. Virginia Stockwell Erwinia amylovora 1 to 2 μm long rod-shaped bacterial cells

Fire Blight  Most severe bacterial disease of pear and apple  First discovered in Hudson Valley – Spread across US, and to Europe, the Middle East and New Zealand  Economically devastating in all areas. Pacific Northwest ○ $89 million (Central WA) ○ $68 million (Hood River Valley) ○ 2002 – Worst epidemic in 100 years Medford, OR

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

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

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 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

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

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 BamHIEcoRI

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

 Locations of orchards with isolates of E. amylovora lacking pEA29 Yakima Valley, WA Sampled orchard Orchard with E. amylovora without pEA29

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

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 Appl. Environ. Microbiol. 70:

 Locations of orchards with isolates that were PCR positive for pEU30 Yakima Valley, WA 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 USDA-ARS, Wenatchee, WA  Krishna Mohan University of Idaho, Parma  Kathleen McNamara Bear Creek Orchards, Medford, OR