ISOLATION OF SPORES FROM ROOT-SOIL MIXTURES

Spores isolated from field-collected root-rhizosphere soil mixtures enable to reveal the spore abundance and species diversity of the arbuscular fungi sporulating in the field. Because a high proportion of arbuscular fungi sporulate seasonally or does not produce spores at all in the field (Brundrett et al. 1999; Stutz and Morton 1996), the determination of the species diversity of arbuscular fungi occurring in a given site needs to conduct long-term and regular sampling of this area, as well as to initiate sporulation of some fungal species in trap cultures. However, Brundrett's et al. (1999) and the author of this website investigations indicated that some species sporulating in the field do not produce spores in trap cultures. Therefore, in ecological investigations, the species diversity of arbuscular fungi of a given site should be determined based on both spores isolated from the field and trap cultures.

Spores from field-collected soil samples, especially those coming from dune soils, usually are highly infective and hence frequently enable to establish one-species cultures, even from single spores. Except for spores of Glomus spp. (many species have ephemeral outer wall layers), most spores of the other genera, especially when recovered from dune soils, contain unchanged diagnostic morphological structures.

Spores of arbuscular fungi are isolated by us using the wet sieving and decanting method described by Gerdemann and Nicolson (1963). The procedure used is as follows:

Step 1. 100 g of air-dried root-rhizosphere soil mixture is placed into a glass container with 1000 ml of tap water. When the mixture contains rough soil, the glass with water and the mixture is kept in a refrigerator at 4oC for at least 12 h.

Step 2. The root-soil mixture is vigorously mixed with a glass rod for 30 sec.

Step 3. After 10-second pause enabling to settle heavier particles and organic material, the remaining soil-root-hyphae-spore suspension is slowly poured through a set of two sieves. The sieves used are those with pores of diameters of 0.5 (the top one) and 0.045 mm. Most spores retain on the 0.045 mm sieve. The top sieve isolates large sporocarps and spores associated with roots.

Step 4. The extracts are washed away from the sieves to Petri dishes of a diameter of 10 cm.

Step. 5. Using a dissecting microscope, spores, aggregates, and sporocarps are picked by means of pipette or needle.

When the root-rhizosphere soil mixture contains a large amount organic material, the isolation of spores is preceded with density gradient centrifugation (Furlan et al. 1980).