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An Improved Diffusion Assay for Quantifying the Polygalacturonase Content of Erwinia Culture Filtrates. Raymond J. Taylor, Postdoctoral research assistant, Department of Plant Pathology, North Dakota State University, Fargo 58105; Gary A. Secor, Associate professor, Department of Plant Pathology, North Dakota State University, Fargo 58105. Phytopathology 78:1101-1103. Accepted for publication 21 March 1988. Copyright 1988 The American Phytopathological Society. DOI: 10.1094/Phyto-78-1101.

The agar diffusion procedure for quantifying pectolytic enzyme activity was modified to optimize assay sensitivity and simplify its implementation. The revised assay was run in 100 50 mm petri plates containing 20 ml of 1% agarose (Type II), ammonium oxalate (0.5%), and sodium azide (0.2%) in phosphate buffer (0.2 M, pH 5.3) with polygalacturonic acid (0.01%) as the substrate. Samples (35 μl) were pipetted into 4.1 mm diameter wells punched in the agarose with a #1 cork borer. After incubation at 37 C for 17 hr, the gel was developed with 10 ml of 0.05% ruthenium red for 30 min, and the diameter of the clear zone of activity was measured microscopically. Polygalacturonase equivalents as low as 2.3 10-4 units were detected. The modified assay required less sample and reduced the problem of gel dehydration associated with the standard assay.

Additional keywords: cup plate assay, pectinase.