A Simple Dual Stain for Detailed Investigations of Plant-Fungal Pathogen Interactions

Dramatic increase in confocal microscopy observation output has been gained by optimization of a simple trypan blue and aniline blue dual-stain and its application to two model pathosystems: Pseudoperonospora cubensiscucumber and Phytophthora infestans-tomato. Comparison of two dual-stain methods for confocal microscopy studies of P. cubensis-challenged cucumber leaves indicated the 'mild' approach most successful. This methodology provides simultaneous detection of different pathogen structures layered with the plant defense reactions. Moreover, ImageJ-assisted quantification of plant defense responses renders this method useful for addressing the host plant resistance reactions, as well as investigating the given isolate's pathogenicity. Application of this method for the P. infestans-challenged tomato leaf samples resulted in detection of several fungal infection structures, along with plant defense responses. The dual-stain also enabled detection of a peculiar aniline blue-sensitive material in the pathogen cell walls at the area of its hyphae emerging through the leaf stomata. Results presented herein indicate this method is applicable for detailed (possibly quantitative) investigations of multiple plant-fungal pathosystems.