kittolab

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:

  • Species-specific

  • Ultra-sensitive

  • Detects teliospores

  • Quick and Simple

  • Applicable for on-site screening of wheat

 

Schematic Representation of Karnal bunt Detection with a Dipstick Immunoassays:

 

  • 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.

 

 

  1. Detection is the primary means of controlling the spread of karnal bunt; no resistant wheat cultivars exist and fungicides are ineffective

  2. U.S. is the worlds leading exporter of wheat - Produces 1/3 of worlds wheat, $4 billion annually

  3. In 1996 and 1997 Karnal bunt was detected in U.S. wheat:

  • Demand and value of U.S. wheat decreased

  • 50 countries refused to accept U.S. wheat.

 

 

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

 

How Do Antibodies Detect Karnal Bunt?

 

 

Results from the Karnal Bunt Immunoassay :

 

The Karnal bunt microwell, sandwich ELISA:

  • Detects as little as 50 ng/well of Karnal bunt protein extract.

  • Works with concentrations of Karnal bunt protein extract between 500ng/well-50ng/well.

  • Distinguishes Karnal bunt from all other fungal contaminants grains.

 

Enhancing Karnal Bunt Detection By Modifying Immunoassay Format:

 

 

Significance of Whole Spore Detection:

  • KB specific antibodies recognize spore wall

  • Disruption of spores not required

    • No denaturing buffer

    • No expensive equipment for mechanical disruption

    • No time-consuming protein extraction steps

  • Immunoassays is cheaper.

     

 

 

 

 

Dipstick Immunoassays detect both Karnal bunt teliospores and protein extract:

 

Dipstick immunoassays are:

  • Quick-Completed within 15 minutes

  • Simple-Consist of only 4 steps

  • Based on visual observation

  • Practical on-site diagnostic tools

Summary:

 

Karnal bunt Immunochemical Techniques:

  • Karnal bunt specific antibodies devoloped

  • Microwell, Sandwich ELISA detects 50 ng of crude Karnal bunt protein extract

  • Luminescent microwell assay detects 100 pg of crude Karnal bunt protein extract

  • Immunoassays detect whole Karnal bunt teliospores. No protein extraction required.

  • Simple easy dipstick format developed, completed within 15 minutes

  • Immunomagnetic isolation ELISA detects 3 spores/mL of suspension buffer.

Future Directions:

  • Develop a 1-step immunomagnetic immunoassay, utilizing colored immunobeads, for detection of Karnal bunt.

  • Devel a dipstick immunoassay tht detects and distinguished multiple grain contaminants

  • Develop immunochemical techniques for rapid detection of Citrus Variegated Chlorosis, the citrus disease caused by Xylella fastidiosa.

 

Acknowledgements:

  • This work has been supported in part by the U.S. department of Agriculture

  • We owe special thanks to LLoyd Wendell and Paul Parker at the Mission, Texas APHIS labaratories of the USDA

  • Laurene Levy, Arnold Tschanz and Lisa Castlebury at the USDA/APHIS, Beltsville, MD

  • Doug Luster at the USDA/ARS, Forst Detrick, MD

  • Blair Goates at the USDA/ARS, Aberdeen, ID

  • Jim Lemburg at the University of Texas at Austin.