",Lethal" Gamma Irradiation

of R. stolonifer, even after a radiation exposure (LDgg) SOMMER, N. F. (University of California, Davis), MIN capable of inactivating the colony-forming ability of the T. CREASY, E. C. MAxIE, AND R. J. RoMANI. Production fungus. This was considered evidence that maceration was Of pectolytic enzymes by Rhizopus stolonifer continuing, even after most fungal cells supposedly had spores after "lethal" gamma irradiation. Appl. Mirogi been inactivated. Therefore, the present study was made 11:463-466. 1963.-Irradiated sporangiospores of Rhizopus to determine the capability of irradiated fungal cells to stolonifer excreted pectolytic enzymes, which hydrolyzed produce enzymes which could macerate plant tissue after pectin and macerated potato tuber discs, into the susdestrution of their colony-forming potential. pending medium. Pectin glycosidase, but not pectin MATERIALS AND METHODS methylesterase, activity developed regardless of the amount of radiation the spores had received, unless the Sporangiospores. R. stolonifer cultures were grown m dose exceeded about 1 megarad. The ability to produce 300-ml Erlenmeyer flasks on 100 -ml of cherry or V-8 juice pectolytic enzymes was found to be more radiationagar medium, and sporangiospores were harvested as resistant than the potential for colony formation or the previously described (Sommer et al., 1963). The freshly ability to germinate. Spores made incapable, through harested sporangiospores were suspended in deionized irradiation, of forming colonies continued to produce ditilled water prior to irradiation. To inhibit germination, pectolytic enzymes after a 6-day period following irradiathe spores were concentrated in the suspension to about tion treatment. 15.5 X 106 spores per ml. Under these conditions, most nonirradiated spores became swollen in a manner typical of the initial stages of germination, but germ-tube forma---Rhizopus stolonifer (Ehr. ex Fr.) Vuill., a black bread tion could be prevented for several days (Sommer et al., mold, is a cosmopolitan fungus capable of attacking and 1963). decaying numerous organic materials, particularly those Irradiation. The irradiation was performed in an AEC of plant origin. The decay in plant tissues is associated CoI0 food irradiator (Romani et al., 1962) at 275 or 320 with, and possibly dependent upon, maceration by hykilorads/hr, depending upon the chamber used. The drolysis of the insoluble pectate of the middle lamella. irradiation temperature was about 27 C. Also, certain fruit hosts of this fungus undergo softening Pectolytic enzyme production. After irradiation, the during ripening as a result of pectin hydrolysis. Prespores were centrifuged, and the irradiated water was sumably, in this latter instance, the fruit tissues are removed and replaced with an equal volume of sterile synthesizing the same or similar pectin-hydrolyzing potato-dextrose-pectin broth medium consisting of 0.8% enzymes as are synthesized by R. stolonifer. Thus, pectoDifco potato extract, 2% dextrose, and 1 % citrus pectin, lytic enzyme production is important both in decay and adjusted to pH 6.5. Portions (20 ml) of the suspension in normal physiological ripening processes of fruit. were placed in 125-ml Erlenmeyer flasks and incubated Several extracellular enzymes are produced by R. at about 24 C on a rotary shaker which provided a swirling stolonifer (Srivastava, Echandi, and Walker, 1959). Pectin motion of 160 cycles/min at an amplitude of about 0.5 in. methylesterases catalyze the hydrolysis of methyl ester At the completion of each 24-hr period, ending at 24, groups of pectin or pectinic acids. Pectin glycosidases, 48, and 72 hr after irradiation, the spores were removed which attack the glycosidic linkages of the pectin chain, from suspension by centrifugation. The liquid portion was catalyze the formation of monogalacturonic acid and collected and held in the frozen state, and the spores were polygalacturonic acids of intermediate molecular weights resuspended in fresh medium after every 24-hr period (Kertesz, 1951). Groups of enzymes, rather than sinle until termination of the test. This potato-dextrose-pectin F0tities, evidently are involved (Saito, 1955; Deuel and broth,containing substances excreted by spores during utz, 1958). one 24-hr period, constituted the pectolytic enzyme ISn unreported work, the authors noticed in strawberry source. fris the extension of water-soaked areas around lesions Tissue maceration. Potato tuber tissue was the substrate 463 on Jne 1, 2017 by gest ht://aem .sm .rg/ D ow nladed fom