6 Wood Discoloration

38 2 Biology
to conserved rDNA regions allows the ITS amplification from fungi without
prior knowledge of their ITS sequence. The PCR products are then digested
either as single or as double digest by restriction endonucleases, of which some
hundreds of different enzymes are known and each having an own recognition
site on the DNA.
Jonsson et al. (1999) identified mycorrhizal fungi in a spruce forest by ITS-
RFLP comparison to reference material. Similarly, Johannesson and Stenlid
(1998) identified tree parasites like Armillaria borealis and Heterobasidion annosum
from bore core samples or mycelia isolated from wood. Regarding wooddecay
Basidiomycetes, Zaremski et al. (1999) differentiated single isolates of
C. puteana, Gloeophyllum trabeum and Oligoporus placenta by ITS-RFLPs.
Various isolates of the closely related S. lacrymans and S. himantioides exhibited
a distinct fragment profile for both fungi after digestion with HaeIII/TaqI
(Schmidt and Moreth 1999). Restriction with TaqI differentiated S. lacrymans;
S. himantioides, D. expansa, C. puteana, A. vaillantii, O. placenta and Gloeophyllum
sepiarium by specific fragments (Fig. 2.21).
Obviously, ITS-RFLP analysis is able to separate various wood decay fungi.
It also detected misnamed isolates assumed to be S. lacrymans (Horisawa et al.
2004; also Fig. 2.21) and identified unknown samples. The method is currently
to be intended as a database for the identification of wood decay and associated
fungi (Zaremski et al. 1999; Adair et al. 2002; Diehl et al. 2004; Råberg et al.
2004).
Advantageous is that the technique is fast and inexpensive. Limitations are:
First, the use of universal primers implies sensitivity to contamination. Second,
Fig.2.21. Species-specific ITS-RFLP pattern of isolates of Serpula lacrymans (L), S. himantioides
(H), Coniophora puteana (C), Donkioporia expansa (D), Antrodia vaillantii (A),
Oligoporus placenta (O), and Gloeophyllum sepiarium (G) generated by TaqI. M marker
(50–1,000 bp). The culture X had been assumed to be C. puteana and was later identified
by sequencing as C. olivacea
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