Traditional Methods

 

Determination keys and descriptions for Deuteromycetes are based on morphology, 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 parameters (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; ). Specific reactions to temperature  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  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 . 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 resources comprising 499 culture collections from 65 countries.

 

 

Table 2.7. Examples of institutions for identification, deposition, and purchasing of microorganisms

 

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  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)
Molecular Methods

 

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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