title: Comparison of cultural and analytical methods for determination of aflatoxin production by Mississippi Delta Aspergillus isolates.
abstract: This study compared cultural and analytical methods for detecting aflatoxin production by Aspergillus species. Aspergillus isolates were obtained from various Mississippi Delta crops (corn, peanut, rice, cotton) and soils. Most of the isolates (99%) were A. flavus and the remainder comprised A. parasiticus and A. nomius. The following three cultural methods were evaluated on potato dextrose agar: fluorescence (FL) on beta-cyclodextrin-containing media (CD), yellow pigment (YP) formation in mycelium and medium, and color change after ammonium hydroxide vapor exposure (AV). Aflatoxins in culture extracts were confirmed by thin-layer chromatography (TLC) and quantified by enzyme-linked immunosorbent assay (ELISA). Of the 517 isolates, 314 produced greater than 20 ng/g of total aflatoxin based on ELISA, and 180 produced greater than 10 000 ng/g of aflatoxin in the medium. Almost all the toxigenic isolates (97%) were confirmed by TLC as producers. Of the toxigenic isolates, as determined by ELISA, 93%, 73%, and 70% gave positive FL, YP, and AV responses, respectively. Of the 203 isolates producing less than 20 ng/g of aflatoxin, 20%, 6%, and 0% of respective FL, YP, and AV methods gave false-positive responses. The 9% false-positive results from TLC fall within this range. This study showed good agreement among all tested cultural methods. However, these cultural techniques did not detect aflatoxin in all cultures that were found to produce aflatoxins by ELISA, LC/MS, and TLC. The best results were obtained when the AV color change and CD fluorescence methods were used together, yielding an overall success rate comparable to TLC but without the need for chemical extraction and the time and expense of TLC.
authors.affiliation: Crop Genetics and Production Research Unit, Stoneville, MS 38776, USA. habbas@ars.usda.gov
authors.forename: Hamed K
authors.initials: HK
authors.lastname: Abbas
authors.forename: R M
authors.initials: RM
authors.lastname: Zablotowicz
authors.forename: M A
authors.initials: MA
authors.lastname: Weaver
authors.forename: B W
authors.initials: BW
authors.lastname: Horn
authors.forename: W
authors.initials: W
authors.lastname: Xie
authors.forename: W T
authors.initials: WT
authors.lastname: Shier
mesh_terms: D000348:Aflatoxins / Q000096:biosynthesis*; D064753:Ammonium Hydroxide; D010367:Arachis / Q000382:microbiology; D001230:Aspergillus / Q000254:growth & development* / Q000302:isolation & purification / Q000378:metabolism*; D001231:Aspergillus flavus / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003470:Culture Media; D003505:Cyclodextrins / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005188:False Negative Reactions; D005189:False Positive Reactions; D005453:Fluorescence; D006878:Hydroxides / Q000378:metabolism; D008922:Mississippi; D009172:Mycology / Q000379:methods*; D012275:Oryza / Q000382:microbiology; D010860:Pigments, Biological / Q000096:biosynthesis; D012988:Soil Microbiology; D003313:Zea mays / Q000382:microbiology; D047392:beta-Cyclodextrins
chemical_list: D000348:Aflatoxins; D003470:Culture Media; D003505:Cyclodextrins; D006878:Hydroxides; D010860:Pigments, Biological; D047392:beta-Cyclodextrins; D064753:Ammonium Hydroxide; C031215:betadex