ISOLATION OF SPORES OF SYMBIOTIC FUNGI FROM ROOT-SOIL MIXTURES

Spores of arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota isolated from field-collected root-rhizosphere soil mixtures enable to reveal the spore abundance and species diversity of these fungi sporulating in the field. However, 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). Therefore, 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 J. Blaszkowski's, 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 AMF were isolated by us using the wet sieving and decanting method described by Gerdemann and Nicolson (1963). The procedure used was as follows:
Step 1. 100 g of air-dried root-rhizosphere soil mixture was placed into a glass container with 1000 ml of tap water. When the mixture contained rough soil, the glass with water and the mixture was kept in a refrigerator at 4oC for at least 12 h.

Step 2. The root-soil mixture was 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 was slowly poured through a set of two sieves. The sieves used were those with pores of diameters of 0.5 (the top one), and 0.045 mm. Most spores retained on the 0.045 mm sieve. The top sieve isolated large sporocarps and spores associated with roots.

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

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

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

Spores of Complexipes moniliformis and species of the genus Endogone, e. g., E. aurantiaca, were isolated as those of AMF.

REFERENCE

Brundrett M. C., Abbott L. K., Jasper D. A. 1999. Glomalean mycorrhizal fungi from tropical Australia. I. Comparison of the effectiveness and specificity of different isolation procedures. Mycorrhiza 8, 305-314.

Furlan V., Bartschii H., Fortin J. A. 1980. Media for density gradient extraction of endomycorrhizal spores. Trans. Br. Mycol. Soc. 75, 336-338.

Gerdemann J. W., Nicolson T. H. 1963. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans. Brit. Mycol. Soc. 46, 235-244.

Stutz J. C., Morton J. B. 1996. Successive pot cultures reveal high species richness of arbuscular mycorrhizal fungi in arid ecosystems. Can. J. Bot. 74, 1883-1889.