Assessment of fluorescein-based fluorescent dyes for tracing Neotyphodium endophytes in planta.

Fluorescent dyes were assessed for their ability to stain viable hyphae of the fungi Neotyphodium lolii and N. coenophialum, symbiotic endophytes of the Pooideae grasses Lolium perenne and Festuca arundinacea respectively. The fluorescein-based fluorophores; fluorescein diacetate (FDA), 5(6)-carboxyfluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor white M2R, were assessed for staining of endophyte hyphae in vitro from axenic fungal cultures and in planta, including epidermal leaf sheath peels, nodes, ovaries, embryos and meristems. CMFDA produced the greatest intensity of staining of fnngal hyphae and gave excellent contrast in planta compared to the plant cells. Compared to the other dyes, CMFDA was also the least affected by photo bleaching and continued to fuoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed. [ABSTRACT FROM AUTHOR]