Symbiotic Fungi: Principles and Practice (Soil Biology)

126 B. Dreyer and A. Morte
8.3.3 Isolation of Intraradical Fungal Structures
The intraradical fungal structures of the roots are isolated following the protocol
described by Saito (1995), with minor modifications.
The following solutions are prepared:
Enzyme digestion solution:
20 g l 1 cellulase
1gl 1 pectolyase
1gl 1 bovine serum albumin
1 mM dithiothreitol (DTT)
0.3 M mannitol
0.01 M MES-NaOH buffer (pH 5.5)
Washing buffer:
0.3 M mannitol
1 mM DTT
0.01 M Tris-HCl, pH 7.4
Approximately 5 g fresh mycorrhizal roots are harvested and washed first
with water and then with 0.5 mM CaSO4 for several minutes. The roots are cut
into 0.5 cm root segments and incubated under shaking in the enzyme digestion
solution at 30 C for 2 h. The digested roots are collected on a 50 mm sieve and
washed with washing buffer. The root segments are then transferred to tubes with
20 ml washing buffer for tissue homogenization using a Polytron (30 s, lowest
speed). The resulting homogenate is filtered through two layers of cheesecloth, the
filtrate is kept in reserve, and the residue is collected in the tubes and homogenized
again with 20 ml washing buffer. This process of homogenization and filtration is
repeated twice more. The filtrates are combined and centrifuged at 1,000 g for
10 min. The pellet is suspended in 2 ml washing buffer and loaded onto a Percoll
gradient consisting of 40, 20, 15 or 10% Percoll in 0.3 M mannitol, 1 mM DTT and
0.01 M Tris-HCl, pH 7.4. The Percoll gradient is prepared by successive loading of
the Percoll solutions in a 50 ml glass centrifuge tube. The centrifugation is performed
at 430 g for 30 min. The fractions enriched in AM fungal structures are
collected from the interfaces between the Percoll bands with a Pasteur pipette.
These fractions are mixed, diluted with 20 ml washing buffer and centrifuged again
at 1,000 g for 5 min. The pellet is transferred to a 50 mm sieve and washed with
washing buffer before being centrifuged again in 10 ml washing buffer at 1,000 g
for 10 min. The resulting pellet contains the AM fungal structures.
Remark: All steps during the enzyme digestion are performed at 4 C.
The isolated fungal structures can be directly subjected to autofluorescence and/
or SDH-activity staining. The process performed for isolating AM fungal structures
does not presumably affect the enzymatic SDH-activity (Saito 1995; Dreyer
et al. 2006).