Vital fluorescent stains for detection of stress in Pythium ultimum and Rhizoctonia solani challenged with viscosinamide from Pseudomonas fluorescens DR54

Abstract

Viscosinamide from Pseudomonas fluorescens DR54 exerts a biocontrol effect on the plant pathogenic microfungi Pythium ultimum and Rhizoctonia solani. Evidence for physiological changes in these and four other fungi was obtained by vital fluorescent staining with Calcofluor white, Nile red, Syto 13, 3,3′-diheptyloxacarbocyanine, 5-(and-6)-carboxy FDA and 5-(and-6)-carboxy SNARF-1, acetoxymethyl, acetate. Based on studies of the fungi grown on solid agar medium and Calcofluor white staining, all isolates responded to viscosinamide treatment by an increased branching, sometimes including hyphal swelling and an increased septation. Further, the staining pattern indicated changes in the esterase activity, intracellular pH and mitochondrial organization and activity. Several of the fungi showed a reduced Nile red staining indicating a general decrease in hydrophobicity of cellular membranes or cell walls. In contrast, only Pythium spp. were stainable with the nucleic acid stain Syto 13 and showed changes in the nuclei in response to viscosinamide. Finally, it was shown that Calcofluor white, Nile red and Syto 13 could also be used to study fungal stress in situ using soil microcosms. The study demonstrated that combinations of vital fluorescent stains are suitable for detailed studies of fungal stress as induced by, e.g., bacterial antibiotics.