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United States Department of Agriculture

Agricultural Research Service

Research Project: BIOLOGICAL CONTROL OF INSECTS AND MICROORGANISMS TO PREVENT MYCOTOXIN CONTAMINATION

Location: Foodborne Toxin Detection and Prevention

Title: Vitality Stains and Real Time PCR Studies to Delineate the Interactions of Pichia anomala and Aspergillus flavus

Authors
item Hua, Sui Sheng
item Brandl, Maria
item Hernlem, Bradley
item Sarreal, Siov

Submitted to: American Society for Microbiology General Meeting
Publication Type: Abstract Only
Publication Acceptance Date: March 2, 2010
Publication Date: May 1, 2010
Citation: Hua, S.T., Brandl, M., Hernlem, B.J., Sarreal, S.L. 2010. Vitality Stains and Real Time PCR Studies to Delineate the Interactions of Pichia anomala and Aspergillus flavus. American Society for Microbiology General Meeting, San Diego, CA, May 23-27, 2010.

Technical Abstract: The objectives of this study were to probe the effect of the yeast, P. anomala against A flavus by using real time RT-PCR technique and vitality fluorescent stains. Yeast and fungi were inoculated into a 250 ml-flask containing 50 ml potato dextrose broth (PDB) at yeast to fungus (Y : F) ratios of 1:1, 5:1, 10:1, 30:1, and 50:1. Yeast cells and hyphae of A. flavus were separated and harvested by filtering through the Cellector tissue sieve. Fungal hyphae were stained with the fluorescent compounds, FUN-1, DiBAC4(5) and CDFA-AM, then viewed under an epifluorescence microscope. Total RNA from yeast cells were extracted and used for first-strand cDNAs synthesis followed by real time PCR. The genes coding for the cell wall degradation enzymes, exo-'-1, 3 glucanase, PaEXG1and PaEXG2 were chosen for analysis. The data suggest that the yeast might inhibit the ATP system of A. flavus. Both DiBAC4(5) and CDFA-AM vitality stains supported the conclusion that the hyphal membrane lost integrity. Real time PCR on P. anomala cDNA showed that both PaEXG1 and PaEXG2 were upregulated by co-culturing with A. flavus. This may indicate cell wall degradation by yeast as a possible mechanism of inhibition of A. flavus resulting in significant reduction of fungal growth.

Last Modified: 4/19/2014
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