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)-carboxy-fluorescein diacetate (CFDA), 5-chloromethylfluorescein diacetate (CMFDA) and the chitin-binding stain, Calcofluor while 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 fungal 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 fluoresce up to 2 h after initial excitation. None of the fluorescent dyes stained fungal hyphae in seed.
Forage Improvement AgResearch Ltd, Palmerston North, New Zealand. stuart.card@agresearch.co.nz
Stuart D
SD
Card
Brian A
BA
Tapper
Catherine
C
Lloyd-West
Kathryn M
KM
Wright
D060026:Endophytes / Q000737:chemistry* / Q000502:physiology; D005452:Fluoresceins / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D055328:Neotyphodium / Q000737:chemistry* / Q000502:physiology; D006109:Poaceae / Q000382:microbiology* / Q000502:physiology; D013194:Staining and Labeling; D013559:Symbiosis
D005452:Fluoresceins; D005456:Fluorescent Dyes