Symbiotic Fungi: Principles and Practice (Soil Biology)

4 In Vivo Model Systems for Visualisation, Quantification and Experimental Studies 57
Fig. 4.4 Schematic representation of the culture system used to study the extraradical mycorrhizal
network. Plants are sandwiched with germinated spores grown on membranes and maintained in
pots for root colonisation; root-adhering mycelium is plucked from the root system, the plant is
transferred onto a new membrane, maintained in petri dish for at least 1 week and eventually
stained for extraradical mycelium visualisation and quantification
sandwiches by immersion in water and checked for the occurrence of root-adhering
mycelium, which is carefully plucked with forceps under a dissecting microscope.
Thus, the experimental system allows the assessment of growth rate and extent of
ex novo produced extraradical mycelium. The occurrence of mycorrhizal colonisation
is assessed by autofluorescence of intraradical fungal structures in fresh whole
roots mounted in water and observed under a light microscope equipped with
epifluorescence optics by using blue light. Mycorrhizal colonisation may be confirmed
on sample plants by clearing and staining with Trypan blue in lactic acid
(Phillips and Hayman 1970).
The roots of each plant are then placed between two mixed cellulose esters
membranes, transferred into 14 cm diameter Petri dishes containing sterile quartz
grit and maintained in a growth chamber as described above. Seven to 21 days after
removing the external mycelium and transplanting, plants are harvested, the root
sandwiches are carefully opened and roots and extraradical mycelium growing
from the roots on the membranes are stained with Trypan blue in lactic acid
(0.05%). Such staining makes the fine network of hyphae extending from colonised
roots visible to the naked eye (Fig. 4.5a).