OPTIMIZATION OF YEAST BIOASSAY FOR TRICHOTHECENE MYCOTOXINS

An improved disc diffusion type bioassay was developed for T-2 toxin ( T-2), HT-2 toxin (HT-2), diacetoxyscirpenol (DAS), deoxynivalenol (DON ), nivalenol (NIV), neosolaniol (NSL), fusarenon-X (FUS-X), trichothec in (TTN), roridin A (RDA) and verrucarin A (VCA) using the yeast, Kluy veromyces marxianus. Factors such as type of medium, agar volume per p late, preincubation time and temperature, incubation temperature, inoc ulum size and pH had variable, and in some cases a dramatic effect on the sensitivity of the assay. The effect of pH of the assay medium was particularly pronounced. The highest sensitivity was obtained when 6 ml per plate of a tryptic-soy-agar (TSA) medium (pH 7.5) containing 10 (5) CFU inoculum per ml was incubated at 38-degrees-C for 18 h. All of the trichothecenes (TNN) were able to inhibit the growth of the yeast with the detection limit being 0.005, 0.01, 0.02, 0.02, 0.1, 0.5, 10, 10, 10 and 50 mug/ disc for VCA, RDA, T-2, TTN, DAS, HT-2, DON, NSL, FUS-X and NIV, respectively. The detection limits for com and wheat sp iked with T-2 were 0.1 and 0.2 mug/g, respectively. Non-TNN mycotoxins that did not inhibit yeast at a concentration of 200 mug/disc were af latoxin B1 (AFB1), ochratoxin A (OA), citrinin (CT), penicillic acid ( PA), cyclopiazonic acid (CPA), penitrem-A (PTA) and zearalenone (ZEE). These results indicate that disc diffusion assay using K. marxianus u nder optimized conditions provides a sensitive method for the detectio n of low concentrations of several TNN.