Fluorescent microscopic technique for viewing fungi in soil and its application to studies of a fusarium-suppressive soil

Abstract

The study of fungal propagules in soil by conventional light microscopy and staining precedures is difficult due to failure to detect propagules obscured by soil particles. Fluorescence microscopy has been used to view fungal spores in soil (Eren and Pramer, 1967: Tsao, 1970) but treatment and staining of the spores with a fluorescent brightener (FB) was required before their addition to soil. Darken (1964) demonstrated that FB increased the germination of fungal spores and, thus, such treatment could substantially alter subsequent fungal development in soil. We describe a simple technique for staining fungal propagules that, unlike the earlier methods, allows the spores to germinate in soil without the influence of FB. The usefulness of our technique was demonstrated by comparing germination of Fusarium oxysporum Schlecht f. sp. hi (Bolley) Snyd. and Hans. chlamydospores in Fusarium wiltconducive and -suppressive soils with and without iron chelates. We previously suggested (Scher and Baker, 1983) that addition of the chelate ethylenediamine di-ohydroxyphenyl-acetic acid (EDDHA) to conducive soil induced suppressiveness to Fusarium wilt diseases because of the high iron-binding ability of EDDHA, which rendered iron unavailable to the pathogen (Kloepper et al., 1980). In contrast, addition of ethylenediamine tetraacetic acid (EDTA) to suppressive soil nullified suppressiveness by releasing loosely-held iron and thereby making iron more available to the pathogen (Kloepper ef al., 1980; Scher and Baker, 1983). F. oxysporum f. sp. lini was grown on potato dextrose agar for 1 week. Conidia and hyphae were scraped from the surface of the agar, homogenized with water in a Waring Blendor (Model 501 I, Waring Products, New Hartford, CT) for 5 min and sieved (< I mm). Ten ml were mixed into each of three 50 g samples of Fusarium wilt-conducive (Colorado sandy loam) and -suppressive (Salinas Valley, California sandy loam) soils. In some treatments, the chelates EDDHA, EDTA, or their ferric complexes, were added (I mg g-t). The soil was air dried for 2 weeks, moistened with IO ml water 50 gg’ soil and air dried for 2 weeks. At this time most of the hyphal fragments and conidia had lysed or converted to chlamydospores. This procedure resulted in approximately IO6 Fusarium propagules gg’ soil. To study chlamydospore germination, 0.2 g soil subsamples were placed in wells in a plastic multi-well tissue-culture plate. Chlamydospores were stimulated to germinate by the addition of 0.1 ml of an aqueous 0.1% glucose, 0.1% asparagine solution to each subsample (Smith and Snyder, 1971). After 16 h at 25°C chlamydospores were stained for viewing by the addition to each subsample of 0.1 ml of a 0.3%