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, gr owing on membranes in microchambers, by using a combination of time-lapse a nd video-enhanced light microscopy and image analysis. The microchambers al lowed continuous observation of living mycelium over a period of several ho urs during which protoplasm flow and bidirectional movements of cellular or ganelles and particles were monitored in individual hyphae. Growth rate of G. rosea hyphae, calculated 8 days after germination, was 2.64 mum/min. Pro toplasmic flow rate, measured on the basis of the movement of particles, ra nged from 2.98 to 4.27 mum/s in living hyphae of G. caledonium. We showed t hat G. rosea, when growing in axenic culture in the absence of the host, ce ased growth within 8 days of germination and underwent a process of protopl asm 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 livin g hyphae by using microchambers could provide useful information on spatio- temporal dimensions of cellular events occurring in arbuscular mycorrhizal fungi.