Microchambers and video-enhanced light microscopy for monitoring cellular events in living hyphae of arbuscular mycorrhizal fungi

Mycelial elongation and protoplasmic flow rate in vitro were monitored for germinated spores of Gigaspora rosea and Glomus caledonium respectively, growing on membranes in microchambers, by using a combination of time-lapse and video-enhanced light microscopy and image analysis. The microchambers allowed continuous observation of living mycelium over a period of several hours during which protoplasm flow and bidirectional movements of cellular organelles and particles were monitored in individual hyphae. Growth rate of G. rosea hyphae, calculated 8 days after germination, was 2.64 mum/min. Protoplasmic flow rate, measured on the basis of the movement of particles, ranged from 2.98 to 4.27 mum/s in living hyphae of G. caledonium. We showed that G. rosea, when growing in axenic culture in the absence of the host, ceased growth within 8 days of germination and underwent a process of protoplasm retraction from hyphal tips, leading to the formation of empty mycelial segments. A process of resource reallocation was inferred in spores of G. rosea showing multiple germination. Detailed developmental studies of living hyphae by using microchambers could provide useful information on spatio-temporal dimensions of cellular events occurring in arbuscular mycorrhizal fungi.