Production, Purification, and Bioassay of Tentoxin. Sami M. Saad, Graduate Assistant, Department of Plant Pathology, University of Wisconsin, Madison 53706; John M. Halloin(2), and Donald J. Hagedorn(3). (2)(3)Postdoctoral Fellow, and Professor, respectively, Department of Plant Pathology, University of Wisconsin, Madison 53706. Phytopathology 60:415-418. Accepted for publication 1 October 1969. DOI: 10.1094/Phyto-60-415.
The chlorosis-inducing toxin produced by Alternaria tenuis was isolated from mycelia and culture filtrates of an isolate of A. tenuis which is pathogenic on beans (Phaseolus vulgaris). No toxin was produced in 28 days by the fungus grown in still culture at 16 or 36 C, or in shake culture for 28 days at 24 C. Maximum toxin production occurred at 28 C in still culture.
Routine purification procedures suitable for large numbers of toxin samples involved removal of polymers by ethanol precipitation, partitioning between diethyl ether and acidic and basic water solutions, and elution from a silicic acid column with ethyl acetate :acetone:n-hexane (2:1:1). Toxin prepared by this method was spectrophotometrically pure and free of interfering effects, such as stunting and germination inhibition of the bioassay plant, that were noted in crude toxin preparations.
The quantitative bioassay for toxin activity involved spectrophotometric estimation of chlorophyll extracted from cotyledons and hypocotyls of cucumber seedlings (Cucumis sativus) with hot ethanol, and is reliable over a concentration range of 1.5-20 µg/ml of toxin.
Chlorosis was visually detectable in seedlings germinated in continuous light at toxin concentrations as low as 0.2 µg/ml, and reached a maximum at a toxin concentration of 20 µg/ml. Seedlings that germinated 48 hr in the dark prior to illumination showed a fourfold increase in chlorosis at low toxin concentrations, as compared with seedlings germinated under continuous light.