Association between microtubules and symbiotic fungal hyphae in protocorm cells of the orchid species, Spiranthes sinensis

Abstract

Seeds of the orchid species, Spiranthes sinensis (Pers.) Ames, were sterilized and germinated in vitro with the symbiotic fungus Ceratobasidium cornigerum (Bourdot) Rogers. Colonized embryos developed into protocorms and these were examined for changes in microtubule arrays, after initial invasion of fungal hyphae into embryos and during peloton formation and degradation. Methods utilized to detect microtubules included immunofluorescence combined with laser scanning confocal microscopy, conventional transmission electron microscopy combined with morphometric analysis, and immunogold labelling. Microtubules were regularly found in close association with intracellular hyphae and degraded hyphal masses. Cortical microtubules disappear during peloton formation but reappear in cells that show fungal lysis. With conventional transmission electron microscopy and immunogold labelling the microtubules associated with fungal hyphae and degenerated hyphal masses were located close to the perifungal membrane that separates fungal hyphae from protocorm cytoplasm.