Submitted to: Canadian Journal of Microbiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/7/2004
Publication Date: 4/20/2004
Citation: Abbas, H.K., Zablotowicz, R.M., Weaver, M.A., Horn, B.W., Xie, W., Shier, W.T. 2004. Comparsion of Cultural and Analytical Methods for Determination of Aflatoxin Production by Mississippi Delta Aspergillus isolates. Canadian Journal of Microbiology. 50(3): 193-199 Interpretive Summary: The use of cultural methods to assess aflatoxin production by Aspergillus flavus can advance ecological and genetic studies on this fungus, and provide economical food safety assessments for lesser-developed countries. This study analyzed 517 Aspergillus isolates using three cultural techniques: yellow pigment production, color change following exposure to ammonia vapor, and fluorescence on B-cyclodextrin amended media compared to two analytical techniques, enzyme linked immunoassay (ELISA) and thin layer chromatography (TLC). All three cultural assays were 86 to 89% reliable in identifying Aspergillus cultures that produce greater than 20 ppb aflatoxin. In the lower range of the assay (less than 20 ppb aflatoxin), the yellow pigment assay produced more false positives. All three cultural assessments can be combined on one plate assay and help overcome limitations of any one cultural assay. These cultural assays are being used in ecological studies assessing the effects of crop management strategies on populations of Aspergillus in soil.
Technical Abstract: This study compared cultural versus analytical methods to detect aflatoxin production by Aspergillus species. Aspergillus isolates (517) were obtained from various Mississippi Delta crops (corn, peanut, rice, cotton) and soils. Ten standard aflatoxigenic A. flavus isolates were also included in this study as reference strains. Cultural methods included fluorescence on B-cyclodextrin media (FL), yellow pigment (YP), and color change after ammonia vapor exposure (AV) on potato dextrose agar. Aflatoxins in culture extract were confirmed by TLC and quantified by enzyme-linked immunosorbent assay (ELISA). Most of the isolates (99%) were A. flavus and the remainder A. nomius and A. parasiticus. Almost all isolates (>90%) producing greater than 20 ppb aflatoxin in the ELISA assay were confirmed as positive producers by TLC analysis. Of the 517 isolates, 314 produced greater than 20 ppb aflatoxin based on ELISA; 89%, 89% and 86% of these isolates gave positive FL, YP and AV responses, respectively. Of the 203 isolates producing <20 ppb aflatoxin, 27%, 46%, and 25% gave positive FL, YP and AV responses. This study showed good agreement between FL and AV methods. However these cultural techniques did not detect aflatoxin in all cultures that were found to produce aflatoxin by ELISA, LC/MS and TLC. Based upon LC/APC/MS and TLC analysis, aflatoxin-positive A. flavus isolates only produced AFB1 or AFB1 and AFB2, while A. nomius and A. parasiticus produced four aflatoxins: AFB1, AFB2, AFG1, and AFG2. These cultural methods can be used for screening of potent aflatoxin-producers, but are not 100% reliable in detecting all aflatoxin-producing Aspergillus species.