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Techniques

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.