Submitted to: Plant Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/21/2004
Publication Date: 6/1/2004
Citation: Pataky, J.K., Block, C.C., Michener, P.M., Shepherd, L.M., Mcgee, D.C., White, D.G. 2004. Ability of an elisa-based seed health test to detect erwinia stewartii in maize seed treated with fungicides and insecticides. Plant Disease. 92:S56 Interpretive Summary: Stewart's bacterial wilt is a common corn disease in the U.S. caused by a bacterium named Erwinia stewartii. Stewart's wilt can be a problem for seed companies who want to export corn from the U.S. because many countries require that imported corn seeds be certified as free from E. stewartii. A lab test called an ELISA is used to test seed for E. stewartii. The ELISA depends on a specific antibody that recognizes only E. stewartii. Seed are blended in liquid buffer and a small amount of the extract is added to a test well on an ELISA plate. After incubation, positive wells are identified as those with enough bacteria to cause a color change from clear to yellow. The amount of color, or intensity, can be measured with a plate reader. A threshold level of color is used to distinguish negative from positive wells. Phytosanitary regulatory agencies in some states have required that all corn seeds submitted for testing be free of chemical coatings. The concern was that seed treatment insecticides or fungicides might interfere with the test sensitivity. Because seed corn is routinely treated with chemicals, companies had to save untreated representative samples from every seed lot they might want to export. This research was done to see if any of the common seed treatment chemicals actually did interfere with the ELISA. The chemicals evaluated included: Actellic, Apron, Captan, Cruiser, Gaucho, Maxim, Poncho, Thiram, and Vitavax in 11 combinations. Extensive lab tests were done to look for large or subtle effects of the seed treatment chemicals. We found that the treated corn seeds did not affect ELISA sensitivity. These results provided evidence that supported the decision by Iowa officials to drop their requirement for untreated seed for phytosanitary testing. They are also being used to support change at the national level. These results provided evidence that supported the decision by Iowa Department of Agriculture and Land Stewardship officials to drop their requirement for untreated seed for phytosanitary testing. They are also being used to support change by USDA Export Services at the national level.
Technical Abstract: Two sets of experiments were done to examine if seed treatment chemicals affected the ability of an ELISA-based assay to detect Erwinia stewartii. The chemicals included Actellic, Apron, Captan, Cruiser, Gaucho, Maxim, Poncho, Thiram, and Vitavax in 11 combinations. In one experiment, treatments were evaluated quantitatively, using seed samples spiked with known amounts of E. stewartii. The numbers of bacteria detected were estimated from ELISA absorbance values using standard curves. Standard curves were created from each ELISA plate using 12 doubling dilutions of a bacterial suspension. Log CFU values for E. stewartii were not significantly different among the untreated control and the 11 seed treatments (P=0.05). Means of log CFU/ml for all treatments were tightly clustered around 5.70, which corresponded to an absorbance value of about 0.440 and a population of about 500,000 CFU/ml. In the second experiment, treatments were evaluated qualitatively. Test samples were created by mixing infected seed lots of Jubilee sweet corn and dent corn inbred A632 with non-infected seed of the same seed treatments so that 100-kernel samples theoretically contained 2-3% infected kernels. Based on binomial probabilities, a few samples were expected to contain no infected kernels and be negative by ELISA. If more than the expected number of negatives were found, a seed treatment would be suspect. The only treatment with a higher than expected number of negatives was A632 treated with Captan/Vitavax. Because Jubilee seed of the same treatment and combinations of Captan/Vitavax with either Apron or Thiram showed no differences, we considered this as a likely Type I experimental error. There was no consistent evidence from either the qualitative or quantitative experiments to suggest that any of the seed treatments affected the sensitivity of the ELISA.