Enzymic diagnosis of copper deficiency in subterranean clover. II. A simple field test

Abstract

A rapid, simple and robust field test is described for diagnosing copper deficiency in subterranean clover plants by measuring ascorbate oxidase activity in young folded leaf blades (YFL) homogenized in phosphate buffer. The test measures activity by counting the drops of iodine required to titrate, to a dark blue end-point, excess ascorbic acid added to and incubated for 20 min with a YFL homogenate. When reagent control titrations had titres of 11 drops of iodine, YFL homogenates from copper-adequate plants had titres of 2-3 drops, from copper-deficient plants 6-11, and from plants with marginal copper supply 4-5 drops. The test was standardized against the measurement of ascorbate oxidase activity in YFL by oxygen uptake. Ascorbate oxidase activity was remarkably insensitive to assay temperature, decreasing by only one-third with decreasing temperature from 30 to 10¦C. It was also very stable in both homogenates and whole leaves. At room temperature, activity dropped by only 25% in homogenates after 6 h and in whole leaf blades after 48 h. When stored in ice, leaf blades retained full activity for at least 5 days. Diagnosis of copper deficiency by the new test agreed closely with diagnosis based on copper analysis of young open leaves taken from the same subterranean clover plants in field pastures. The test should allow extension workers to give on-the-spot advice about the copper status of pastures containing subterranean clover.