Relation of Polyphenol Oxidases to Fungitoxicity.

Stemphylium sarcinaeforme (Cav.) Wilt. (formerly Macrosporium sarcinaeforme Cav.) and Monilinia fructicola (Wint.) Honey (formerly Sclerotinia fructicola (Wint.) Rehm.) are two fungi which have been widely used as test organisms for screening fungicides in the laboratory. S. sarcinaeforme is resistant to the toxicity of phenolic and quinonoid compounds (herein referred to as phenols and quinones) while M. fructicola is not. S. sarcinaeforme is typically dark colored, while M. fructicola is hyaline or buff-colored. As dark pigmentation is frequently associated with polyphenol oxidase activity,1 it was postulated that S. sarcinaeforme must have an active polyphenol oxidase which could oxidize and polymerize phenols and quinones, thus rendering them innocuous to the organism. Polymerization would be an essential part of the detoxification because the mere oxidation of a phenol would probably produce a toxic quinone. It was further postulated that M. fructicola probably lacks an active polyphenol oxidase that could detoxify these compounds, or else contains, in vivo, an inhibitor of the system; e.g., an active hydrogen donor.2 In order to explore these hypotheses, it was first necessary to ascertain what type of polyphenol oxidase, if any, was present in each fungus. The next step was to determine the relationship between fungitoxicity of phenols and quinones and their resistance to being changed by enzymes found in the fungi. Methods.-The fungi were grown in still culture on casamino acid media,3 S. sarcinaeforme for 3 weeks and M. fructicola for 2 weeks. Once the mycelial mats were harvested, all succeeding steps for extraction were done at 0° to 5°C. The harvested mats were washed, broken up in a Waring Blendor with 50 cc. of distilled water, and then ground to a cell-free preparation using a tissue homogenizer aided by the addition of powdered alumina. This material was dialyzed overnight and then centrifuged at 18,000 r. p. m. for 1/2 hour. The supernatant was used as the enzyme preparation for the tests to be described. The activity of the mycelial extract was determined by mixing it with aqueous solutions or suspensions of the phenols or quinones being tested. The basic mixture was 1 cc. of a 1.0%o solution or suspension of the test compound, 1 cc. of pH 5.0 buffer and 0.1 cc. of the mycelial extract. A change in color indicated that the test compound was being acted upon by the enzyme(s) present in the extract. Absence of color change indicated that the compound was not oxidized and polymerized. If polyphenol oxidases were present in the mycelial extracts, failure to give a color reaction with a given compound could result from two causes: (a) the compound is not a substrate for the enzyme, or (b) the compound is an inhibitor of the enzyme. These two possibilities were distinguished by replacing 1 cc. of the buffer with 1 cc. of a known substrate, catechol, at 0.01 AM in pH 5.0 buffer. If the catechol in this mixture did not darken, it was concluded that the test compound was an inhibitor of the enzyme. For each compound, four test combinations were