Leaf-clearing technique to assist fungal spore germination counts.

Abstract

FOR comparisons between formulations of certain fungicides, it was necessary to estimate the percentage germination of Alternaria solani and Venturia inaequalis spores inoculated on to sprayed tomato and apple leaves. Direct microscopic examination was generally difficult owing to the opacity of the leaf. When the leaves were cleared by immersion in solvents such as pyridine1 or dioxan and propionic acid2, the results were misleading because many of the spores were washed off. Pressing leaves on to gelatin or cellulose acetate3 on slides and removing them proved time consuming, and often failed to remove all of the spores. Moreover, debris and the impression of the leaf surface made it difficult to decide whether many of the spores had germinated. Stripping of a cellulose acetate film4,5 from the leaves and dissolving it in acetone on a slide was an improvement, but nevertheless tedious. As direct examination of the spores in situ would be the most rapid method of estimating the percentage germination, a technique was developed which allows accurate and rapid sporecounts on large numbers of leaves.