Epifluorescence microscopy imaging of phytoplasmas in embedded leaf tissues using DAPI and SYTO13 fluorochromes

The use of DNA-specific dyes, i. e. DAPI, is extensively reported for phytoplasma detection in fresh plant materials. However, fluorescence-based microscopy and imaging of fresh tissues often evidences technical limitations which are more significant in infected tissues, because phenolic and other defense-related compounds accumulate in the cell wall and in the vacuole making difficult sample preparation. In this paper we describe a method based on the use of epifluorescence microscopy and the DNA probes DAPI and SYTO13® for phytoplasma visualization in resin-embedded plant tissues. The method allows detection of phytoplasmas and it is recommended for tissues that are recalcitrant to conventional imaging.