Symbiotic Fungi: Principles and Practice (Soil Biology)

12 Interaction with Soil Microorganisms 203
and candicidin. The antibiotics, glucose, magnesium chloride, and TS were sterilized
by filtration through a 0.2 mm membrane and added separately to autoclaved
and cooled CMS before the plates were poured. The solidified medium was covered
by a cellulose ester membrane (mesh size 0.3 mm; Millipore). Soil samples (0.5 g)
were suspended in 100 ml sterilized, distilled water and vigorously shaken for
30 min. The membrane surface was inoculated with 200, 100, and 50 ml aliquots.
This approach selects for actinomycetes; only these are able to penetrate the
membrane pores in order to grow into the underlying agar. In contrast, growth of
other bacteria is restricted to the membrane surface. The purity of optically distinguishable
colonies was verified by repeated culture of diluted samples on ISP 2 agar
in the dark at 20 C (Shirling and Gottlieb 1966).
12.5 Procedures
12.5.1 Culture on Solid Media
For testing the effect of bacteria on fungal growth, dual cultures were used. The
fungal inoculum was excised from the actively growing edge of a fungal colony
using the wide end of a Pasteur pipette and transferred to the center of a sterile
cellophane sheet (exclusion limit 10 kDa; Folia, Wendelstein, Germany) on top of
ISP 2 agar and contained in a 9-cm-diameter Petri dish (compare Fig. 12.1;
Maier 2003). Bacterial isolates were applied to the edge of the Petri dish outside
the cellophane layer as a thin line of about 4 cm in length. The distance between
the inocula was approximately 3.5 cm. In order to provide sufficient gas
exchange, the Petri dishes were not sealed with Parafilm. Contamination was kept
to a minimum by placing the Petri dishes in miniature plant culture chambers at
20 C in darkness. For preliminary screening, two independent trials with four
parallels each were performed. With bacterial isolates which had been shown to
promote or suppress fungal growth, culture experiments were extended for up to 11
weeks (slow growing Amanita muscaria) with three trials each consisting of 20
parallels.
Stock solutions of bioactive secondary metabolites from AcH 505 culture
supernatant, the growth-promoting compound auxofuran and antifungal compound
WS-5995 B (Riedlinger et al. 2006), were prepared by dissolving the substances in
MeOH at concentrations of 50 mM for auxofuran and 30 mM for WS-5995 B.
7-dehydroxyauxofuran, which is liquid at room temperature, was diluted to obtain a
concentration of 50 mM in MeOH. After a short incubation at room temperature,
the stock solutions were vortexed vigorously to ensure that the compounds were
completely dissolved before use. For all substances, concentrations ranging from
150 nM to 150 mM were used to assess their impact on fungal growth.
For the analysis of mycelial growth on solid media, Petri dishes were filled with
35 ml MMN medium. The compounds were added to 40 C warm medium in 100 ml