Quantification of mycelium of Botrytis spp. and the antagonist Ulocladium atrum in necrotic leaf tissue of cyclamen and lily by fluorescence microscopy and image analysis

A technique was developed to localize and quantify the internal mycelial co lonization of necrotic leaf tissue of cyclamen (Cyclamen persicum) or lily (Lilium) by pathogenic Botrytis spp. and the antagonist Ulocladium atrum. T his technique allows investigation of competitive substrate colonization by both fungi, which is a key process for biological control of Botrytis spp. by U. atrum. A combination of differential fluorescent labeling and image analysis was applied on cryostat sections of necrotic leaf tissue. Botrytis mycelium was labeled specifically by indirect immunofluorescence using a m onoclonal antibody specific for Botrytis spp. and an antimouse fluorescein conjugate. Wheat germ agglutinin conjugated to the fluorochrome TRITC was u sed to label mycelium of both fungi. Image analysis was used to measure the relative surface area of the cryostat section covered by fluorescing hypha e of Botrytis spp. and by fluorescing hyphae of both fungi. A mathematical conversion was derived and used to calculate the relative mycelial volume o f each fungal species in the necrotic tissue based on the measured relative surface areas. Temporal aspects of substrate colonization were studied in a short time series. An analysis of components of variance provided insight into spatial colonization patterns for the fungal species involved and all owed the design of efficient sampling strategies for future experiments.