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Immunoassays For Fungal
Disease Of Wheat
Developing Diagnostic
Tests To Protect U.S. Agriculture
What are we detecting?

Objective: Develop a diagnostic tool that will detect low levels of
Karnal bunt, a smut fungus that infects wheat.
Approach:
Develop an immunoassay that is:
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Schematic Representation of Karnal bunt Detection with a Dipstick
Immunoassays:
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- Fungal pathogen of wheat
- Causal organism: Tilletia indica
- Lessens both quality and quantity of
wheat
- Karnal bunt is established in: India,
Afghanistan, Lebanon, Pakistan, Iraq, Turkey, Syria, Sweden,
Mexico.
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Detection is the primary means
of controlling the spread of karnal bunt; no resistant wheat cultivars
exist and fungicides are ineffective
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U.S. is the worlds leading
exporter of wheat - Produces 1/3 of worlds wheat, $4 billion annually
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In 1996 and 1997 Karnal bunt was detected in
U.S. wheat:
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How do we make antibodies specific for
Karnal Bunt?

- Produce polyclonal antibodies against crude
KB extract
- Pour anti-KB serum over a Tb/Tt protein
extract affinity column
- Collect antibodies from wash step
- Subtractive hybridization produces KB
specific antibodies
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How Do Antibodies Detect Karnal Bunt?

Results from the Karnal Bunt Immunoassay :

The Karnal bunt microwell, sandwich ELISA:
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Detects as little as 50 ng/well of Karnal bunt
protein extract.
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Works with concentrations of Karnal bunt protein
extract between 500ng/well-50ng/well.
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Distinguishes Karnal bunt from all other fungal
contaminants grains.
Enhancing Karnal Bunt Detection By Modifying
Immunoassay Format:

Significance of Whole Spore Detection:
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Dipstick Immunoassays detect both Karnal bunt
teliospores and protein extract:
Dipstick immunoassays are:
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Quick-Completed within 15 minutes
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Simple-Consist of only 4 steps
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Based on visual observation
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Practical on-site diagnostic tools
Summary:
Karnal bunt Immunochemical Techniques:
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Karnal bunt specific antibodies devoloped
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Microwell, Sandwich ELISA detects 50 ng of crude
Karnal bunt protein extract
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Luminescent microwell assay detects 100 pg of crude
Karnal bunt protein extract
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Immunoassays detect whole Karnal bunt teliospores.
No protein extraction required.
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Simple easy dipstick format developed, completed
within 15 minutes
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Immunomagnetic isolation ELISA detects 3 spores/mL
of suspension buffer.
Future Directions:
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Develop a 1-step immunomagnetic immunoassay,
utilizing colored immunobeads, for detection of Karnal bunt.
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Devel a dipstick immunoassay tht detects and
distinguished multiple grain contaminants
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Develop immunochemical techniques for rapid
detection of Citrus Variegated Chlorosis, the citrus disease caused by
Xylella fastidiosa.
Acknowledgements:
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This work has been supported in part by the U.S.
department of Agriculture
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We owe special thanks to LLoyd Wendell and Paul
Parker at the Mission, Texas APHIS labaratories of the USDA
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Laurene Levy, Arnold Tschanz and Lisa Castlebury at
the USDA/APHIS, Beltsville, MD
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Doug Luster at the USDA/ARS, Forst Detrick, MD
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Blair Goates at the USDA/ARS, Aberdeen, ID
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Jim Lemburg at the
University of Texas at Austin.
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