Submitted to: American Chemical Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 4/30/2002
Publication Date: 6/2/2002
Citation: LAO, Y., ABBAS, H.K., SHIER, W.T. 2002. THE YELLOW PIGMENTS USED IN THE RAPID IDENTIFICATION OF TOXIGENIC STRAINS OF ASPERGILLUS FLAVUS ARE AFLATOXIN BIOSYTNTHETIC INTERMEDIATES. AMERICAN CHEMICAL SOCIETY ABSTRACTS. Vol. 39:56. Interpretive Summary:
Technical Abstract: Aflatoxins (AF), among the most potent carcinogens known, are produced by some, but not all, strains of Aspergillus flavus and A. parasiticus. The higher percent toxigenic strains in the soil reservoir of A. flavus than in naturally-infected crop plants lends support to strategies for reducing AF contamination by preinoculation with non-toxigenic strains. Saito and Machida (Mycoscience, 40, 205, 1999) developed a rapid method for identifying AF-producing and non-producing strains of A. flavus and A. parasiticus, which may provide a useful pre-screen for identifying non-toxigenic strains. In this method, the reverse side of colonies of AF-producing strains on potato dextrose agar (PDA) medium turn from yellow to pink immediately after exposure to ammonium hydroxide vapor. A. flavus cultures on PDA were lyophilized, extracted with methanol until no more yellow pigment was obtained, and the extracts combined and evaporated. The yellow pigment is a pH indicator dye, which turns from yellow to pink at pH greater than or equal to 8 by addition of any base, and turns yellow again when the pH is lowered with acid. The methanol extract was dissolved in water, applied to an open C18-silica reversed phase column and eluted with a methanol step gradient. Pigments (14) which changed color (usually yellow to pink) when spots on TLC plates were exposed to ammonium hydroxide vapor, were isolated by repeated preparative normal phase TLC on silica gel. Seven pigments representing most of the color, were identified by comparing UV spectra, negative ESI-MS/MS and 1H-NMR properties with literature or predicted values. All are known intermediates in the biosynthesis of AF: versicolorin C, versicolorin A hemiacetal, nidurufin, averufin, versicolorin A, norsolorinic acid, and averantin. Seven more pigments with the same UV profile, which could not be identified as known AF biosynthetic intermediates, were isolated in amounts too small to permit structure identification; additional yellow pigments were present, but not obtained pure. Identification of the pigments that predict AF production by A. flavus strains as being AF biosynthetic intermediates provides a convenient rationalization for the predictive power of the method. Strains producing pigment amounts below the level of visible detection would be expected to give false negative results. Strains with a mutation in a biosynthetic enzyme beyond that which makes norsolorinic acid would be expected to give false positive results.