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

Hua S. T., Brandl M., Hernlem B. J., Sarreal S. L., Lee B.

Author address: 

USDA-ARS, Western Regional Res. Ctr., Albany, CA.


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-β -1, 3 glucanase, PaEXG1and PaEXG2 were chosen for analysis. Collected fungal hyphae were then rinsed with distilled water, transferred to a pre-weighed filter paper and dried in an oven at 65°C overnight for biomass determination of A. flavus. Fluorescent dyes that measure different cellular physiological states are useful for a comprehensive understanding of the metabolic state of the cells. The FUN-1 fluorescent stain binds to nucleic acids. Biochemical processing of the dye by metabolically active fungal hyphae yielded cylindrical intra-vacuolar structures (CIVS) that were markedly red shifted in fluorescent emission and spectrally distinct from the nucleic acid bound form of the dye, which results in green fluorecence. The formation of CIVS is mediated by ATP production. Metabolically active A. flavus hyphae accumulated red fluorescence in vacuoles, wheras hyphae that were inhibited by P. anomala stained green. The number of CIVS in the hyphae was greatly reduced in the dual culture, which suggested 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 integity. Real time PCR on on P. anomala cDNA showed that both PaEXG1 and PaEXG2 of P. anomala were upregulated by co-culturing with A. flavus. This may indicate cell wall degradation by yeast is a possible mechanism of inhibition of A. flavus.

abstract No: 


Full conference title: 

110th General Meeting American Society for Microbiology
    • ASM 110th (2010)