6 Wood Discoloration

36 2 Biology
single strands at about 94 ◦ C (denaturation), two nucleotide primers (15–30
bases) attach to the complementary nucleic acid region at 35–60 ◦ C (annealing),
and a thermostable polymerase synthesizes two new single strands at
about 72 ◦ C (extension) by starting at the primers and using the four nucleotides
present in the reaction mixture (Mullis 1990), that is the target DNA
is doubled with each cycle.
In real-time PCR techniques, the accumulation of PCR product is detected
in each amplification cycle either by using a dye or a fluorescently labeled
probe. Hietala et al. (2003) quantified Heterobasidion annosum colonization in
different Norway spruce clones using multiplex real-time PCR. Eikenes et al.
(2005) monitored Trametes versicolor colonization of birch wood samples. The
technique of PCR-DGGE was used for arbuscular mycorrhizal fungi. A nested
PCRofvariableregionsofthe18SrDNAwascombinedwithsubsequent
separation of the amplicons using denaturing gradient gel electrophoresis
(DGGE), and the method is intended to be used to discriminate closely related
Glomus species (Vanhoutte et al. 2005). Vainio and Hantula (2000) performed
DGGE analysis of fungal samples collected from spruce stumps.
Randomly amplified polymorphic DNA (RAPD)-analysis
RAPD analysis is based on PCR, but uses only one, short (about ten bases)
and randomly chosen primer, which anneals as reverted repeats to the complementary
sites in the genome. The DNA between the two opposite sites with
the primers as starting and end points is amplified. The PCR products are
separated on agarose gels, and the banding patterns distinguish organisms
according to the presence/absence of bands (polymorphism). It is a peculiarity
of RAPD analyses that they discriminate at different taxonomical level, viz.
isolates and species, depending on the fungus investigated and the primer
used (Annamalai et al. 1995).
RAPD was used for tree parasites, such as Armillaria ostoyae (Schulze et al.
1997) and H. annosum (Fabritius and Karjalainen 1993; Karjalainen 1996),
mycorrhizal fungi (Jacobson et al. 1993; Tommerup et al. 1995) and edible
mushrooms (Lentinula edodes: Sunagawa et al. 1995). Regarding wood decay
fungi, Theodore et al. (1995) showed for S. lacrymans polymorphism among
eight isolates. Another RAPD analysis exhibited similarity within S. lacrymans,
which may be attributed to the low genetic variation of the species, but “normal”
polymorphism in S. himantioides and Coniophora puteana (Schmidt and
Moreth 1998).
The German isolate Eberswalde 15 of C. puteana is obligatory test fungus for
wood preservatives according to EN 113. The isolate is known for its variable
behavior in wood decay tests. RAPD analysis was able to show that some alleged
Ebw. 15 cultures held in different test laboratories are in reality subcultures
from the British facultative test isolate FPRL 11e (Göller and Rudolph (2003),
which explains the varying test results.
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