Symbiotic Fungi: Principles and Practice (Soil Biology)

24 Auxin Production by Symbiotic Fungi 389
24.4.5 Estimation of the IAA Amount Produced by the Fungus
Establish a dose response of A. thaliana to synthetic IAA as described below.
Prepare IAA standard solutions in ethanol, e.g. 1 mM, 10 mM, 100 mM. Apply
45 ml of the solution per filter disc (FD). Place the FDs on aluminum foil in a
cleanbench, and allow the solvent to evaporate completely (this may take a few
hours). Prepare FDs with pure solvent (ethanol) for the control Petri dishes.
Place the FD on the agar as indicated in Fig. 24.1 (2 FDs per Petri dish), add the
seeds of the test plants as described earlier and grow the plants for ten to 15 days.
Compare the morphological changes to the rooting system induced by the fungus to
changes caused by known IAA amounts.
Remark: Alternatively, auxin may be added directly to the medium. In this case,
a stock solution in ethanol is prepared, and a suitable volume is added to the
medium after autoclaving when the medium has cooled down to a temperature of
about 60 C. Mix by swinging the bottle gently. Instead of IAA, the more stable
derivative a-naphtalene acetic acid (NAA) may be used (Fig. 24.2f), but care must
be taken when working with auxin deficient mutants. NAA may enter the roots by
diffusion, circumventing carrier mechanisms (Delbarre et al. 1996).
Remark: Fungal culture filtrate may also be used directly or after extraction
with ethyl acetate to test the presence of IAA. In the case of Piriformospora,
inoculate an appropriate medium, e.g. 100 ml M+ (4 g l 1 yeast extract, 10 g l 1
malt extract, 4 g l 1 glucose, 20 g l 1 ), with 20 mycelial plugs from the growing
margin of a colony on solid medium. Mock-inoculate control flasks with plugs from
solid medium without fungus. Be sure to use medium prepared the same day and
stored under the same conditions for fungal cultures and controls. In a first experiment,
cultures should be harvested at different time points, e.g. once a week (at least
one fungal culture and one control), until the timepoint of maximal IAA concentration
is known. Separate mycelium from medium by filtration through filter paper.
Take 15 ml of the culture supernatant and of sterile medium as a control. Sterilize 5
ml by sterile filtration, apply to filter paper discs as described above (in experiments
with culture filtrate of Piriformospora indica, 135 ml per disc were effective).
Adjust the remaining 10 ml of medium to pH 3 with acetic acid, and extract with
equal volume of ethyl acetate. Evaporate the organic phase to dryness under
vacuum or nitrogen stream. Resolve the residue in 200 ml of ethyl acetate, apply
to filter paper discs (45 mL on one disc) and repeat the bioassay.
Remark: The bioassays described above should not be considered as a proof
that a fungus produces IAA, because other substances may cause similar effects, or
inhibiting substances may interfere with the effect of IAA. It rather provides a quick
indication that a fungus produces IAA or metabolite(s) with similar effects.
24.4.6 IAA Quantification in Agar Plates
Slope agar plates or dual bioassay plates are prepared as described above. Three
Petri dishes are inoculated with the fungus (Piriformospora or truffle) as described