Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides ): Comparison of Quantitative Brine Shrimp Assay with Physicochemical Analysis

Abstract

Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides ) produce T-2 toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox medium. At 15 and 27°C, maximum T-2 toxin yield (265 and 50 μg/ml) was found after 10 to 14 and 7 days, respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27°C and slowly at 15°C. Addition of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15°C (400 μg of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes, however, requires a more time-consuming chemical analysis.