A KOH-aniline blue technique for fluorescent staining of fungi in association with plant tissues was developed. The technique provided rapid, simple, and effective documentation of plant-fungal interactions with specimens representing the Deuteromycotina, Ascomycotina, Basidiomycotina, and Mastigomycotina. Applications of the technique were investigated, including documentation of host-penetration events, characteristics of host colonization, fungal reproduction, and detection of inoculum. In the standard KOH-aniline blue procedure, fresh specimens were autoclaved for 15 min at 121°C in 50 ml of 1 M KOH, rinsed in deionized water, mounted in the stain solution, and examined with ultraviolet fluorescence. The stain solution was prepared as 0.05% aniline blue dye (CI #42755 or CI #42780) in 0.067 M K 2 HPO 4 at pH 9.0. Modifications of the standard procedure also were tested, including use of variously preserved specimens, alteration of the KOH treatment, and moderate variation of the stain solution. The technique produced a high degree of resolution and contrast between hyphae and host-plant tissues. The resulting documentation supported previous observations and, in some cases, provided new information about the nature of specific host-pathogen interactions.
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Michael E. Hood (1996) studied this question.