Technique for visual demonstration of germinating arbuscular mycorrhizal spores and their multiplication in pots

Abstract

We describe a simple technique for the germination of arbuscular mycorrhizal (AM)–fungal spores and their multiplication in pots. Glomus fasciculatum, G. mosseae, and Gigaspora margarita were used. A single wheat seedling was tied to a glass slide, previously covered with filter paper with the help of thread. One single surface‐sterilized AM‐fungal spore was placed on the middle portion of the root of the wheat seedling using a sterilized syringe. The slide was placed vertically in a 100 mL glass beaker filled with 25 mL of root exudates–water (1:4, v/v) solution, which was collected by growing twenty wheat seedlings in a 150 mL beaker filled with 100 mL sterilized distilled water for 7 d. The slide was observed daily using a compound microscope to follow the time course of germination. In this technique, the spore is directly in contact with the host root, and a visualization of spore germination, hyphal development, and appressorium formation is possible without disrupting fungal growth or the establishment of the symbiosis. The method allows to document the germination events and to assess hyphal‐elongation rates by photographing the same spore on consecutive days. The inoculated seedling was used to initiate single‐spore multiplication in a sterilized (autoclave on 3 alternate days at 120°C for 120 min at 1.05 kg cm–2 pressure) potted sandy soil (150 mL volume) into which the slide with the inoculated seedling was inserted carefully through a previously made slit. The wheat seedlings in all pots (4 treatments and 15 replications) became colonized by mycorrhiza, confirming that the establishment of the AM‐fungal symbiosis is highly reproducible. Our technique permits the relatively undisturbed growth of the symbiotic partners, the visualization of germinating AM‐fungal spores, and their multiplication in pots. This simple and low‐cost method facilitates the production of pure lines of AM fungi from single spores, allowing for the study of intraspecific variation and potentiality for cytological, biochemical, physiological, and taxonomical studies.