Symbiotic Fungi: Principles and Practice (Soil Biology)

118 H. Dupré de Boulois et al.
7.6.2 Adding the Isotopic Tracer
After the addition of medium in the HC, extraradical hyphae will develop rapidly
and extend in the whole compartment. Generally, within 1–4 weeks the density of
the ERM will be sufficient to expose it to the isotopic tracer. However, it is also
possible to expose the ERM to the labelled medium contained in the HC as soon as
it crosses the partition wall (see Table 7.3).
The isotopic tracer can be added to the HC either directly in/on the medium
contained in this compartment depending on whether liquid or solid medium is
used, or after renewing it. Note that if the isotopic tracer is added on solid medium,
its diffusion into the medium should be verified (using autoradiography for
instance, Gray et al. 1995). Renewing liquid medium is done by gently removing
the old medium using a pipette and adding new medium. In the case of solid
medium, a scalpel and forceps are used to cut and remove the medium. Thereafter,
new medium is added in the HC. Solid medium should be cooled at a temperature of
40 C before its addition in order to reduce the risk of damaging the roots and AM
fungi. The temperature of the liquid medium should be 25–27 C.
Renewing solid medium in the HC means that the AM fungal mycelium growing
in this compartment will be removed. However, rapid development of AM fungal
mycelium is usually observed. This operation might be even useful to synchronize
ERM growth in the HC.
It should be noticed that it is also possible to mix the isotopic tracer directly with
the medium before addition in the HC. For some experiments, for instance on C
transport, the isotopic tracer should be placed in the RC. As the medium cannot be
removed, the isotopic tracer must then be added on the solidified medium.
To get a more accurate measurement of the transport and metabolism processes,
it can be useful to suppress from the medium the element (or a chemical analog)
under study. For instance, Declerck et al. (2003) strongly reduced the concentration
of K in the RC and had no K in the HC to study Cs transport. Identically, Jin et al.
(2005) used medium lacking N but amended with 15 N-labelled substrates to study N
transport and metabolism in AM symbiosis. However, it should be taken into
consideration that removing or modifying the concentration of one element
from the culture medium will perturb the concentration of at least one other element
and the balances between elements. This may therefore have an impact on the
growth of the AM fungus and host.
7.6.3 Exposure Time
There is no predefined time for contact between the ERM and the isotopic tracer.
AM transport and biosynthetic pathways vary depending on the element or substances
under study but also on the fungal strain, age of the culture and ERM
density in the HC. In Table 7.3, a few representative examples are given for five
isotopic tracers and with the four major techniques used to detect them.