Symbiotic Fungi: Principles and Practice (Soil Biology)

278 P.A. Olsson
Furthermore, C translocation by mycelium themselves can be estimated and the
sampling is easier when mycelium is growing in liquid medium. Mycelia and roots
are stored at 20 C until analysed for 13 C enrichment and fatty acid content and
composition.
16.3.3 Labelling in Pot Experiments
Controlled labelling of experimental pots can be obtained by transferring them to a
greenhouse seedling propagation box (volume around 25 dm 2 ). The transparent lid
of the greenhouse box can be adapted with a fan, a thermometer, and a gas-sampling
tubing connected to an infra-red gas analyser (IRGA) and a small hole with a
septum. The bottom tray and the lid of the box are sealed with paraffin and the
initial CO2 concentration recorded. Labelling is done when the fan is turned on by
injection of, for example, 100 ml of 13 CO2 (99% 13 CO2) with a gas-tight syringe
through the septum. The CO 2 concentration inside the box increases usually from
240–270 to 500–1,000 ppm after such an injection. A pulse period of 2–4 h is
usually appropriate. We have observed that assimilation may be very low the first
hour after labelling, and therefore a longer period than 1 h is recommended. It is
best to investigate the assimilation using the IRGA. The lid is removed after the
labelling time.
16.3.4 Labelling in the Field
13 C labelling in the field can be done by enclosing a piece of vegetation, or a single
shoot, in a plastic chamber and then injecting the labelled gas. Basically the
labelling in the field works in the same way as for pot cultures. Almost all
incorporation goes via the autotrophic shoots. The chamber may first be emptied
for non-labelled CO 2, which can be done by letting the plants themselves empty
CO2 in an enclosed room. Usually carbon dioxide content goes down rapidly after
enclosing in a closed chamber. The initial carbon dioxide, and increase due to
labelling, can be measured using an IRGA coupled to the chamber. Each system is
unique and it is wise to make a pilot study for each type of plant, or plant species.
The fraction of C that moves below ground to the roots may in some cases be small
and a rather high label in roots is needed to be able to track the C further to AM
fungi and rhizosphere bacteria. The initial labelling can be checked by sampling
shoots directly after the pulse period. If the labelling is too small, it is very difficult
to trace the C later on after the chase period. If the initial labelling results in a delta
13 C value of around 1,000 (corresponding to around 2% 13 C, which means around
1% enrichment), there is a good chance of tracing the C further. If it is around delta
100, the chances are small.