Symbiotic Fungi: Principles and Practice (Soil Biology)

56 M. Giovannetti et al.
4.2.4 Remarks
The methods described allowed the visualisation, for the first time, of anastomoses
in different species of the genus Glomus. Time-course experiments showed that
hyphal tips were able to fuse with hyphae growing nearby in about 35 min, and that
a bidirectional flow of particles (vacuoles, mitochondria, nuclei, and fat droplets)
moved at the speed of 1.8 0.06 mm s 1 through hyphal bridges formed during
anastomosis (Giovannetti et al. 1999, 2000). Protoplasmic continuity, the characteristic
feature of successful hyphal fusions, was evidenced by the complete disappearance
of hyphal walls and visualised by histochemical localisation of formazan
salts in hyphal fusions, after SDH staining. The established protoplasmic flow was
further demonstrated by the detection of nuclei in hyphal bridges, evidenced by
DAPI staining. Moreover, the described methods revealed that interactions between
hyphae of species of the genera Gigaspora and Scutellospora never led to anastomosis
formation (Giovannetti et al. 1999). This evidence was confirmed by other
authors in in vitro monoxenic cultures (de la Providencia et al. 2005).
The described methods allowed the detection of nonself incompatibility in presymbiotic
mycelial networks of AMF (Giovannetti and Sbrana 2001; Giovannetti
et al. 2003). These findings, suggesting that AMF can recognise self-entities
and discriminate self from nonself, opened the way to vegetative compatibility
tests, already used for the identification of genetically different isolates of pathogenic,
saprophytic and ectomycorrhizal fungi (Fries 1987; Sen 1990; Leslie 1993;
Dahlberg and Stenlid 1994; Cortesi et al. 1996; Milgroom and Cortesi 1999;
Glass et al. 2000). Such tests, carried out on geographically different isolates of
Glomus mosseae, showed that hyphal interactions between different isolates never
produce anastomoses, suggesting their genetic isolation (Giovannetti et al. 2003).
4.3 Visualisation and Quantification of Intact Mycelial
Networks Spreading from Mycorrhizal Roots
4.3.1 Experimental System
This experimental model system has been devised for visualising and quantifying
intact extraradical mycelia growing from mycorrhizal roots into the surrounding
environment (Fig. 4.4). Surface-sterilised seeds of experimental plants are germinated
in moist sterile quartz grit. After 15 days the root system of each seedling is
sandwiched between mixed cellulose esters membranes containing germinated
spores (obtained as described in Sect. 4.2). The seedlings with the sandwiched
root systems are placed into pots filled with sterile quartz grit. Pots are closed in
transparent polyethylene bags and maintained in a growth chamber with 24 C day
and 21 C night temperature and 16/8 light/dark cycle. After 30 days’ growth in the
sandwich system, the plants are harvested, the roots are gently removed from