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United States Department of Agriculture

Agricultural Research Service

Research Project: Control of Aflatoxin Production by Targeting Aflatoxin Biosynthesis

Location: Food and Feed Safety Research

Title: Testing the efficacy of eGFP-transformed Aspergillus flavus as biocontrol strains

Authors
item Moore, Geromy
item Mack, Brian
item Beltz, Shannon

Submitted to: Food and Nutrition Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 22, 2013
Publication Date: April 30, 2013
Citation: Moore, G.G., Mack, B.M., Beltz, S.B. 2013. Testing the efficacy of eGFP-transformed Aspergillus flavus as biocontrol strains. Food and Nutrition Sciences. 4:469-479.

Interpretive Summary: A method of pre-harvest biological control against aflatoxigenic fungal contamination in agricultural commodities involves field inoculation with non-aflatoxigenic Aspergillus flavus. Yet to be studied are the long-term impacts of annual applications of these “stable” strains. This manuscript is the initial study to develop a means of tracking the movement and persistence of biocontrol strains that have been tagged with green fluorescent protein. Laboratory investigations included monitoring potential impacts of the GFP on colony growth and fluorescence, followed by homologous and heterologous comparisons of strains to determine any potential impact of GFP on the ability of biocontrol strains to compete against both atoxigenic and toxigenic wild type strains. Overall there is no difference in growth of wild type and transformed strains, and their fluorescence shows promise for future studies using GFP-tagged biocontrol strains.

Technical Abstract: Current biological control methods to prevent pre-harvest aflatoxin contamination of corn, cottonseed, and ground and tree nuts involve field inoculation of non-aflatoxigenic Aspergillus flavus. To date, the efficacy of this approach requires annual reapplication of the biocontrol agent. The reason for this requirement is uncertain. To track the dispersal and test the longevity of these strains, we prepared fluorescent biocontrol strains by incorporating into them the gene expressing the enhanced green fluorescent protein (eGFP). We first investigated the effects of eGFP transformation on the ability of the fluorescent fungus to compete with its non-fluorescent homolog, and then with other heterologous non-aflatoxigenic strains as well as with aflatoxigenic isolates. Our findings indicate that, in these studies, detection of fluorescence was variable, with some fluorescent strains exhibiting enhanced growth and sporulation post-transformation. In our tests, not all transformed strains proved to be good candidates for tracking because their fluorescence was reduced over the course of our study. Most of the transformed strains retained fluorescence and showed robust colony growth in an artificial competitor environment; therefore, they should be suited for further trial under more natural settings. Our ultimate objective is to determine if out-crossing between biocontrol strains and native field populations is occurring in a natural setting.

Last Modified: 7/28/2014