ARS | Publication request: Vitality Stains and Real Time PCR Studies to Delineate the Interactions of Pichia anomala and Aspergillus flavus

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.