Enzyme-Linked Immunosorbent Assay for Plant Viruses in Intact Leaf Tissue Disks

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Abstract
Tobacco ringspot virus (TRSV) and maize dwarf virus (MDMV) were detected by enzyme-linked immunosorbent assay (ELISA) when leaf [corn and geranium] tissue disks were substituted for leaf extracts as the test sample. The procedure entailed incubating 6 mm diameter disks of leaf tissue, sampled with a paper punch, in microtiter plate wells containing 200 .mu.l of phosphate-buffered saline with Tween 20 and polyvinyl pyrrolidone. When a single virus-infected leaf disk was assayed, the absorbance (A405 nm) obtained was poorly reproducible, ranging 0-50% of that observed by testing the leaf extract. A more comparable test reliability was obtained by increasing the 2 h substrate reaction time to 24 h and by incubating several leaf disks in each plate well. By using this protocol, MDMV in corn and TRSV in geranium were detected from 1 wk up to at least 2 mo. postinoculation with a reliability comparable to the conventional test using a leaf extract. The leaf disk sampling technique, when used to complement the ELISA test, should prove valuable for the rapid and sensitive qualitative evaluation of virus infection when dealing with large numbers of plants.