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ARS Home » Southeast Area » Stoneville, Mississippi » Crop Genetics Research » Research » Publications at this Location » Publication #168581

Title: Yellow Pigments Used for Rapid Identification of Aflatoxin-producing Aspergillus Strains are Anthraquinones Associated with the Aflatoxin Biosynthetic Pathway

item SHIER, W.
item Abbas, Hamed

Submitted to: Bioorganic Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/6/2005
Publication Date: 10/13/2005
Citation: Shier, W.T., Lao, Y., Steele, T.E., Abbas, H.K. 2005. Yellow Pigments Used for Rapid Identification of Aflatoxin-producing Aspergillus Strains are Anthraquinones Associated with the Aflatoxin Biosynthetic Pathway. Bioorganic Chemistry. 33:426-438.

Interpretive Summary: Cultural methods used for assessing aflatoxin-producing potential in isolates of the common food and crop contaminant fungus Aspergillus flavus are important tools for monitoring food contamination in developing countries. They are also useful in studies aimed at identifying A. flavus strains that do not produce the harmful toxin where these strains could protect the crops from being infected by harmful fungal isolates. The present study identified the molecular mechanisms of two potentially useful cultural tests for toxin production in fungus. In each of these cultural assays, pigments produced by toxic fungus are detected either directly on the undersides of the fungus itself or by inducing a color change in the media on which the fungus grows. The pigments formed in the two assays are chemical components used by the fungus to manufacture the toxins, which for some reason are produced in larger amounts than needed for production of the toxins. Recognizing the molecular mechanisms of these tests make it possible to predict the conditions under which false test results occur. It is expected that understanding the molecular mechanisms of these assays will encourage their use in research and in the monitoring of crops for food safety.

Technical Abstract: Various approaches are being studied to reduce aflatoxin contamination, including biological control by applying non-toxigenic Aspergillus flavus to crops before they become colonized by toxigenic strains from the soil reservoir or other sources. These studies have created a need for rapid, reliable methods to prescreen isolates for aflatoxigenicity. We have evaluated two empirical cultural methods for predicting aflatoxigenicity: yellow pigment production and color change after exposing cultures to ammonium hydroxide vapor. Yellow pigments in aflatoxigenic A. flavus cultures and in extracts from them behaved like pH indicator dyes turning plum-colored on addition of base, and reverting to yellow on acidification. Thirteen pigments have been isolated from the extract by reverse phase chromatography on C18-silica followed by preparative normal phase thin layer chromatography on silica gel using color changes on exposure of chromatography spots to ammonium hydroxide vapor to guide fractionation. The structures of seven pigments have been established to be norsolorinic acid, averantin, averufin, versicolorin C, versicolorin A, versicolorin A hemiacetal and nidurufin using spectroscopic methods and comparison of chromatographic characteristics to published values. All are known anthraquinone intermediates on the aflatoxin biosynthetic pathway, except nidurufin, which is a known anthraquinone product of a branch pathway in Aspergillus spp. When cultures of a non-aflatoxigenic A. flavus isolate were extracted and fractionated in an identical manner, none of the identified anthraquinone pigments could be detected by HPLC. These observations support the validity of the yellow pigment and ammonia vapor methods for predicting aflatoxigenicity, and predict limited numbers of false positives or false negatives.