Determination keys and descriptions for Deuteromycetes are
based on mor-phology, color, and development (conidiogenesis) of conidia and
conidio-genous cells (Carmichael et al. 1980; Domsch et al.
1980; v. Arx 1981; Wang 1990; Hoog and Guarro 1995; Schwantes 1996; Kiffer and
Morelet 2000; Samson et al. 2004). The fruit bodies of Ascomycetes and
Basidiomycetes serve to identify species on the basis of macro- and microscopic
characteristics using keys or illustrated books: Kreisel 1961; Domariski 1972;
Domariski et al. 1973; Breitenbach and Kranzlin 1981, 1986, 1991, 1995; Moser
1983; Jiilich 1984; Hanlin 1990; Jahn 1990; Wang and Zabel 1990; Ryvarden and
Gilbertson 1993, 1994; Huckfeldt and Schmidt /005; yeasts: Barnett et al.
1990).
There are identification kits for yeasts that employ assimilation tests
of carbohydrates with a specifically adapted database, and also growth tests on
carbon sources that are bound to a tetrazolium dye (Mikluscak and Dawson-Andoh
2005). An illustrated key for wood-decay fungi is in the Internet (Huckfeldt
2002). For wood-inhabiting Basidiomycetes, of which only mycelium is present,
keys are based on microscopic characteristics of the hyphae and on growth
pa-rameters (Davidson et al. 1942; Nobles 1965; Stalpers 1978; Rayner and Boddy
1988; Lombard and Chamuris 1990). Among the physiological characteristics, the
Bavendamm test for the differentiation of brown- and white-rot fungi is based
on the presence/absence of the phenol oxidase laccase (Bavendamm 1928; Davidson
et al. 1938; Kaarik 1965; Niku Paavola et al. 1990; Tamai and Miura 1991; Chap.
4.5). Specific reactions to temperature (Chap. 3.4) provide further
information. However, keys for mycelia are unable to differentiate closely
related fungi such as the various Antrodia and Coniophora species. The strand
diagnosis of Falck (1912; Table 2.4, Figs. 8.19-8.21) differentiates few indoor
decay fungi like Serpula lacrymans, Coniophora puteana and Antrodia vaillantii.
As house-rot fungi
are the economically most important wood fungi by destroying wood during its
final use within buildings and as not all indoor fungi fruit, a key including
about 20 strand-forming indoor wood decay fungi (Huckfeldt and Schmidt 2004,
2005, 2006) is given in Appendix 1. In addition, there are monographs and descriptions
of important tree pathogens (e.g., Ceratocystis and Ophiostoma species:
Upadhyay 1981; Wing-field et al. 1999; Armillaria species: Shaw and Kile 1991;
Heterobasidion annosum: Woodward et a1.1998) and of wood-degrading
Basidiomycetes (Cockcroft 1981; Ginns 1982) with data to taxonomy, morphology,
ecology, growth behav-ior, and wood degradation in the laboratory and outside.
A further possibility for identification is by national
institutions against fee (Table 2.7). A list of collections and institutions
with strain collections, compiled by German Collection of Microorganisms and
Cell Cultures, is in the Internet (www.dsmz.de/species/abbrev.htm). Sixty-one
culture collections in 22 Eu-ropean countries are united in the European
Culture Collections' Organisa-tion (ECCO; eccosite.org). The World
Federation of Culture Collections is a
worldwide database on culture re-sources comprising 499 culture collections
from 65 countries.
Table 2.7.
Examples of institutions for identification, deposition, and purchasing of
mi-croorganisms
German Collection of Microorganisms and Cell Cultures
(DSMZ), Braunschweig Centraalbureau voor Schimmelcultures (CBS), Baarn,
Netherlands International Mycological Institute (IMI), Kew, UK Belgian
Coordinated Collections of Microorganisms (BCCM), Gent American Type Culture
Collection (ATCC), Rockville
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 nitrocellu-lose 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 (32P or 35S) showing the hybridized fragment by
autoradiography. Biotin, dioxigenin, or fluores-cein 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 single strands at about
94°C (denaturation), two nucleotide primers (15-30 bases) attach to the
complementary nucleic acid region at 35-60 °C (anneal-ing), and a thermostable
polymerase synthesizes two new single strands at about 72 C (extension) by
starting at the primers and using the four nu-cleotides 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 PCR of variable regions of the
18S rDNA was combined with subsequent 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 com-plementary 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 "nor-mal" 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 which explains the varying
test results.
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