Isozyme characteristics of Caloscyphafulgens infested and pathogen-free spruce seed samples and use of alkaline phosphatase activity for qualitative and quantitative disease incidence assays

Extracts of samples from Caloscyphafulgens infested and noninfested Sitka spruce (Piceasitchensis (Bong.) Carr.) seed lots and diseased and healthy seeds were separated by electrophoresis on polyacrylamide gels and stained for esterase (EC 3.1.1.1), leucine aminopeptidase (EC 3.4.1.1), acid phosphatase (EC 3.1.3.2), alkaline phosphatase (EC 3.1.3.1), glucose-6-phosphatase (EC 3.1.3.9), malate dehydrogenase (EC 1.1.1.37), isocitrate dehydrogenase (EC 1.1.1.42), 6-phosphogluconate dehydrogenase (EC 1.1.1.43) and alcohol dehydrogenase (EC 1.1.1.1). Several differences were detected between the isozyme patterns of disease-free and infested samples, but the main difference was the latter's high alkaline phosphatase activity. By using gel electrophoresis, (i) a qualitative analysis was developed, based on the presence or absence of alkaline phosphatase, to distinguish infested from disease-free seed lots, and (ii) the high alkaline phosphatase activity of infested samples was determined to be of pathogenic origin. By determining the alkaline phosphatase activity of samples with known numbers of diseased seeds, a prediction equation was derived relating enzyme activity and disease incidence. Correlation analyses showed significant (P = 0.01) correlations between disease incidence estimates obtained by this technique and plating of surface-sterilized seeds on water agar.