Understanding Erwinia Amylovora : - PowerPoint PPT Presentation

the causal agent of fire blight of pear and apple n.
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  1. The causal agent of fire blight of pear and apple. Erwinia amylovora1 to 2 μm long rod-shaped bacterial cells Understanding ErwiniaAmylovora: Alyssa Carey Dr. Joyce Loper Dr. Virginia Stockwell

  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

  3. Disease Cycle of Fire Blight Spring Cankers activate, Erwiniaamylovoraspread by bees and rainand grows on flower tissues X Early Summer Infection of blossoms Shoot and fruit infection Winter Pathogen overwintersin cankers Summer and FallPathogen kills branches and forms cankers

  4. Research Opportunities • Periodic fire blight epidemics continue to damage orchards causing great losses • There are few options for disease control • Erwiniaamylovoralong considered homogeneous but recent findings indicate that the pathogen may be more diverse http://www.sorengallery.com/images/ra20gp2040x48.jpg

  5. Summer Research • Characterize isolates of Erwiniaamylovorafrom 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/Eamylovora.gif

  6. pEA29 • Considered near ubiquitous in Erwiniaamylovora. • Associated with virulence/fitness, but not essential • Target for some PCR methods for identification/detection of E. amylovora

  7. Hypothesis • Erwiniaamylovoraisolates from the Pacific Northwest contain extrachromosomal DNA in addition to pEA29

  8. Goals • Isolate and characterize plasmids of Erwiniaamylovorafrom 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

  9. Procedure • Obtain samples from orchards • Dilution series • Plate onto agar medium for growth • Confirm identity as Erwiniaamylovora • PCR reactions • AgriStrip test • Sequencing of 16S rRNA • Isolate plasmid DNA using alkaline lysis method • PCR reactions for pEA29 • Restriction digest or RFLP analysis

  10. 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

  11. 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

  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 http://farm3.static.flickr.com/2324/2065568371_c2bfc1c0b7.jpg?v=0

  13. Yakima Valley, WA • Locations of orchards with isolates of E. amylovora lacking pEA29 Sampled orchard Orchard with E. amylovorawithout pEA29

  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 Erwiniaamylovora EcoRI digest

  15. pEU30 • Another plasmid in Utah strain of E. amylovorafound 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.

  16. Locations of orchards with isolates that were PCR positive for pEU30 Yakima Valley, WA Sampled orchard Orchard with E. amylovorawith pEU30

  17. Current Research Activities • Sequencing an uncharacterized plasmid(s) from my collection • Four isolates with unique RFLP patterns were selected • Isolates were cured ofpEA29 using eviction mutagenesis

  18. Summary • 205 isolates of E. amylovora • Over half had altered plasmid profiles • 6 lacked pEA29 • 27 were positive for pEU30

  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