OCCURRENCE OF A MYCOTOXIN, OCHRATOXIN A, IN WHEAT AND ISOLATION OF OCHRATOXIN A AND CITRININ PRODUCING STRAINS OF PENICILLIUM VIRIDICATUM Ochratoxin A is a metabolite of Aspergillus ochraceus

Ochratoxin A is a metabolite of Aspergillus ochraceus Wilh. (5) and of Penicillium viridicatum Westling (8). It is highly toxic to rats and ducklings and produces liver and kidney damage (6). Both A. ochraceus and P. viridicatum have been isolated from stored grain, and ochratoxin A itself was recently found in corn (7). The presence of this toxin in rolled wheat used as livestock feed is reported here. In addition, two strains of P. viridicatum that produce ochratoxin A were isolated from the wheat. One of these strains also produces citrinin, a nephrotoxin formed by several species of Aspergillus and Penicillium (3). Four samples of moldy red spring wheat, taken from the same heated pile at different times and depths, were forwarded from Canada Department of Agriculture, Calgary. The wheat had become wet while in the granary, and some had been included in the rations of cattle that subsequently died. Each sample was powdered (20 mesh) and 50 g were extracted with 250 ml methanol-water (55:45 v/v) plus 100 min-hexane in a Waring blendor. After centrifugation of the mixture, 50 ml of the aqueous methanol layer were removed by pipette and shaken with two 50-ml portions of chloroform. The combined chloroform extracts were evaporated to dryness on a hot water bath under nitrogen, and the residue was dissolved in 0.5 ml chloroform and refrigerated. Ochratoxin A was detected in the four sample extracts after thin-layer chromatography (TLC) on 0.3 mm layers of Adsorbosil 5 silica gel, using tolueneethyl acetate-90% formic acid (6: 3: 1 vIvIv) for development. Each extract formed a green fluorescent spot under ultraviolet light with the same R r (0.55) as authentic ochratoxin A. The fluorescence turned blue on treatment with ammonia. Amounts of ochratoxin A, estimated by visual comparison of fluorescence intensities of spots from extract and standard, were 20, 100, 85, and 85 ,ug per kg for the four samples. The ochratoxin A in sample 2 (lOa ,ug per kg) was confirmed using two additional solvent systems, benzene-metilanol-acetic acid (24: 2: 1, vIvIv) and benzene-acetic acid (9:1 v/v). Microgram quantities of the toxin were obtained by preparative TLC. It was soluble in sodium bicarbonate solution. The fluorescent methyl ester formed with BCl, or BF3 in methanol corresponded, on TLC, to that formed from authentic ochratoxin A. The method for isolation of fungi from the wheat kernels was based on that of Wallace and Sinha (l 0). Incubation of wheat sample 2 at 25 C and 30 C yielded two unidentified Penicillium spp. and a number of other fungi tentatively identified as Aspergillus glaucus and A. candidus. None of these isolates was found to produce any of the fluorescent mycotoxins, ochratoxin A, citrinin, aflatoxins, aspertoxin, or sterigmatocystin. Incubaticn at low temperature (l2 C), however, led to successful isolation of toxin-producing cultures. After incubation for 2 weeks, abundantly sporulating Penicillium spp. had emerged from the wheat;