provided the wild-type (WT) strain. A suspension containing 1-4 X 10" viable cells/ml was prepared by adding sterile saline (0.85 % NaCl) to a 24-hr culture. To obtain streptomycin-resistant mutants, 10 ml of this suspension were added to a sterile Petri dish, and agitated during irradiation with ultra-violet light (450 ergs/mm^) to yield 5 % survival. The treated suspension was serially diluted and plated on potato-dextrose agar containing 0.5 % yeast extract (PDY) and 30 mg of streptomycin (740 units/mg) per liter of medium. The resistant mutants were serially transferred at least three times on PDY and only those mutants which were still resistant and both virulent and prototrophic were retained. One mutant was designated as the (P) strain and used for further study.Approximately 3 X 105 viable cells/ml of the P strain were suspended in a solution of 1.7 X 10-3 M N-methyl-N'-nitro-N-nitrosoguanidine (NTG) in 0.1 M tris buffer (tris [hydroxymethyl] amino methane), pH 7.0, or phosphate buffer, pH 7.2 for 25 min. at 25 °C. This treatment gave a survival of 25 % of the cells as determined from a standardized dosage-response curve. Single colonies from surviving cells were transferred to PDY in a Petri dish and evenly spaced to form a 26-colony grid. Surface-disinfested celery petioles were inoculated with a 24-hour old colony using a needle to wound the abaxial (inside) surface. From one grid of 26 colonies, 26 sites 1 cm apart were inoculated on the petiole. After incubation for 24 hr at 27 °C in a moisture-saturated chamber, the presence or absence of soft rot at the site of inoculation was scored. Avirulent colonies were transferred four times on PDY and tested for avirulence, prototrophy, and streptomycinresistance after each transfer. Colonies retaining these characteristics after the last transfer were considered to be avirulent (AV) mutants.Cells from one AV mutant were treated with NTG and plated on Difco desoxycholatelactose agar (DOCL). The virulent prototrophic and streptomycin-resistant colonies were termed (RV) mutants. Recommended routine tests (Manual of Microbiological Methods 1957, BuRKHOLDER 1957 were made for the characterization of the wild-type species and the mutant strains (FRIEDMAN 1964) under identical cultural conditions. Stocks of all strains were kept lyophilized in sealed tubes under vacuum until needed. Additional virulence and cultural tests of all strains preceded their use in the enzyme assays.Enzyme assays. Five media were employed for the cultivation of the strains in the different enzyme assays. Each medium was adjusted by adding 0.1 N NaOH to yield a final pH of 6.8 after autoclaving. Erlenmeyer flasks (250 ml) containing 50 ml of medium were inoculated and incubated for 24 hr at 28 °C in a water-bath rotary shaker (K' inch circular orbit, 150 rotations/min.).Medium I. 0.24% KH,PO^, 0.08% CaCl2-2H2O, 0,02% MgSO^VH^O, 0.3% (NH^)2SO^, 0.5 % monosodium glutamate, 0.2 % yeast extract (Difco), 0.4 % polygalacturonic acid (no. 3491, Sunkist Growers, Inc.) and 0.1 % carboxymethylcellu...

Culture filtrates from a virulent and an avirulent strain of each of three phytopathogenic species of Pe?zicilliunz grown in a defined medium to which were added different sources of organic carbon had endopolygalacturonase (endoPG) and exopolygalacturonase (exoPG), but neither detectable pectin methylesterase nor pectin lyase activity. Extracts fro~n diseased tissue of oranges inoculated with P . italicunz or P . digitatzrm and from diseased tissue of apples inocula,ted with P . enpanszrnz, but not from healthy fruits, had endoPG and exoPG activity. Culture liltrates and extracts of diseased tissue were s~~bjected to vertical starch-gel zone electrophoresis. The number, location (anodic, cathodic), electrophoretic mobility, and relative activity of sites of endoPG or exoPG activity were determined by the species, virulence or avirulence of the strain, source of organic carbon, and gel pH.