6 Wood Discoloration

2.4 Identification 35
antibodies. Antisera produced by animals like mice and rabbits as answer
to the injection of mycelial fragments, extracts or culture filtrates are investigated
by Western blotting, enzyme-linked immunosorbent assay (ELISA)
or immunofluorescence (Clausen 2003). However, the experiments often exhibit
cross-reactions with non-target organisms, even when monoclonal antibodies
after fusion with myeloma cells (hybridomas) are used. Investigations
were performed with e.g., Armillaria spp., Coniophora puteana, Gloeophyllum
trabeum, Lentinula edodes, Lentinus lepideus, Oligoporus placenta, Phellinus
pini, S. lacrymans, Trametes versicolor and with wood-stain fungi (Jellison and
Goodell 1988; Palfreyman et al. 1988; Breuil et al. 1988; Glancy et al. 1990;
Burdsall et al. 1990; Vigrow et al. 1991b, 1991c; Clausen et al. 1991, 1993; Kim
et al. 1991a, 1991b, 1993; Toft 1992, 1993; McDowell et al. 1992; Clausen 1997a;
Breuil and Seifert 1999; Hunt et al. 1999).
The diagnostic potential lies in the identification of species without the
need of preceding isolation and pure culturing and in the detection of fungi
at early stages of decay (Clausen and Kartal 2003). The methods may become
applicable when the producing techniques for hybridomas and diagnostic kits
have been established.
Immunological methods were also used to visualize the distribution of
enzymes of wood-degrading fungi within and around the hypha and in woody
tissue (e.g., Kim 1991; Kim et al. 1991a, 1993; Chap. 4).
2.4.2.2
DNA-Based Techniques
Southern blotting of restriction fragments (RFLPs)
In the RFLP technique, nuclear, mitochondrial or chloroplast DNA is treated
with endonucleases, which each have a short nucleotide recognition site on
the DNA target, and which cut the DNA into fragments. The fragments are
separated on agarose gels and transferred by Southern blotting on nitrocellulose
or nylon membranes. The addition of a special nucleotide probe, which
hybridizes with a fragment, selects fragments from the present bulk (“smear”)
of fragments. The probe may be radioactively labeled ( 32 Por 35 S) showing
the hybridized fragment by autoradiography. Biotin, dioxigenin, or fluorescein
probes visualize the fragment colorimetrically or as chemoluminescence.
The different fragment pattern (restriction fragment length polymorphisms,
RFLPs) differentiate species, intersterility groups and isolates, like as it was
used e.g., for Armillaria spp. (Schulze et al. 1995, 1997).
The technique is exact, but needs time and is methodically longwinded.
Methods using the polymerase chain reaction (PCR)
The procedure of PCR multiplies a part of DNA by a repeated (25–40 times)
three-stage temperature cycle (amplification): the double strand is split into its
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