Approaches For Studying The Community

Many studies of wood-decay fungi have been based on what is fruiting on the surface. The advantage of such observations is that a large number of woody units can be surveyed in a relatively short time. Further, fruiting reflects the reproductive output and thereby represents an important aspect of fitness. However, there are several reasons why studies solely based on fruiting structures will only give a partial view of the composition, activity and importance of the fungal community involved in wood decomposition. One reason is that this gives a heavy bias towards species with large conspicuous fruit bodies, in particular basidiomycetes, predominantly belonging to the polyporacae, agaricales and corticiacae. Organisms such as bacteria, arthropods and fungal species lacking large fruit bodies will remain undetected by this approach. Other drawbacks are associated with the timing of fruiting. Inevitably, there is a lag phase between establishment of a mycelium and formation of a fruit body. The time required to build enough resources within the mycelium for fruiting can be from a few weeks up to several years depending on species, resource quality and environmental conditions (Chapter 5). For example, a species that in spite of being established in early stages of decomposition only produces fruit bodies at late stages of the development, will erroneously appear to be a late successional. Other species might have a much shorter lag before fruiting and the interpretation might again be misleading. Furthermore, certain cues might be needed for fruiting, such as specific temperature regimes or light conditions, and surveys during periods outside the required conditions will fail to record them. For species with annual fruit bodies, fruiting might be irregular, so there is a need to revisit sites for comprehensive inventories (e.g. Lindner et al., 2006). Berglund et al. (2005) stated that monitoring should be performed on a stand scale and focus on species with durable fruit bodies, for example polypores. This will provide data that can be used both to detect future changes in biodiversity in old-growth spruce forests and to evaluate conservation strategies.

Another complicating factor is the need for mating to occur before fruit bodies can be produced. This can be seen as an example of the Allee effect, resulting in negative population growth at low densities (Courchamp et al., 1999). This may be misinterpreted as substrate specificity if detection is based solely on fruiting. However, the difficulty in fertilizing homokaryons is a challenge for rare bas-idiomycetes where the low density of airborne spores makes the probabilities for dispersal to new habitats low and the likelihood of two spores reaching the same resource unit even lower. This would be counteracted partly by homokaryotic mycelia defending areas of suitable wood habitat from invasion by other fungi and thereby acting as spore traps over extended periods (Adams et al., 1984; James and Vilgalys, 2001; Edman et al., 2004a, 2004b). However, the dynamics and the genetics of wood colonization has been poorly studied and whether an extended homokaryotic stage is common enough to represent any significant contribution to the basidiomycete life-cycle remains to be proven.

A more direct method for studying decay fungi is to isolate mycelia into pure culture (Table 4). Isolation methods have the potential to be more inclusive than

Table 4 Comparison of different approaches to identification of wood-inhabiting fungi

Table 4 Comparison of different approaches to identification of wood-inhabiting fungi

Method

Strengths

Weaknesses

Opportunities

Threats

Fruiting body

Well-established

Irregularity in

Large scale

Overemphasizing

inventories

method; a large

fruiting; need to

comparative

species groups with

number of resource

revisit sites at

studies and surveys

conspicuous fruit

units can be

different seasons

are possible; species

bodies;

covered in

etc.; inconspicuous

with reliable

misinterpretation of

relatively short

fruiting or non-

fruiting patterns

temporal relations

time; fruiting is a

fruiting mycelia can

(e.g. perennial

good measure of

go undetected; only

polypores) can be

fitness; low cost

the surface is sampled; limited possibility to study functional aspects; requires skills in species identification

used as indicators, also by amateurs

Mycelial

Focus on the active

Time consuming;

Physiological and

Overemphasis on

identification

mycelia in the

requires high skills

genetic

heavily sporulating

resource; relatively

in mycelial

experiments can be

and fast growing

low cost

identification (but can be aided by molecular identification)

made

microspecies; missing non-culturable species

Molecular

Direct identification

Time consuming;

Functional studies

Species can be more

identification

of species present

high cost; requires a

can be made

or less readily

in the substrate;

molecular lab

directly in the

amplified (primer

allows for precise

resource;

bias)

identification

comprehensive sampling of the community

fruit body monitoring although normally they require a higher input of labour. Here the bias is towards species that have a rapid growth and are adapted to growth on artificial media which might not be the same conditions as those for growing in wood (Menkis et al., 2004; Lindahl et al., 2007). For example, heavily sporulating microfungi are typically over represented in isolation work. One important advantage with the isolation approach is that experimental work, for example physiological potential or combative strength (Holmer and Stenlid, 1997; Holmer et al., 1997), can be carried out.

Another approach is to use molecular methods for detection (Table 4). Samples can be taken directly from wood for DNA extraction and subsequent PCR amplification. Typically, ribosomal genes are targeted with the ribosomal internal transcribed spacer region (ITS) giving enough interspecific variation for species identification. In addition, the more conserved large and small subunits give phylogenetic information that are very useful for homing in to the right higher level taxonomic groups. Amplicons can be purified directly from an electrophoresis gel, or cloned into a bacterial library and thereafter se-quenced. Identification is carried out by comparing the sequence of the target gene with databases of known sequences. Public databases such as GenBank have a large number of such sequences but wood-inhabiting fungi are not always comprehensively represented, and therefore, complementary information might be needed from local databases. For ectomycorrhizal fungi, a common database UNITE, is published with quality checked information (Koljalg et al., 2005). Amplification from environmental samples will yield a mix of several amplicons. The challenge then is to be able to detect most members of a fungal community from the mixture of several DNA types. One approach is to study RFLP (Johannesson and Stenlid, 1999) or T-RFLPs (Allmer et al., 2006) involving amplification using marked primers followed by restriction enzyme treatment allowing for separation of individual DNA types. Matching the T-RFLPs with predicted databases after treatment with two or three enzymes allow for species-specific detection. To avoid problems with unpredicted discrepancies from theoretical T-RFLP patterns, the reference library for T-RFLPs should be made from actual restriction cuts of the specimen from clone libraries or isolates from the sampling site. An alternative way to separate the primary amplicons is to use a denaturating or temperature gradient gel electrophoresis, DGGE (Vainio and Hantula, 2000) and TGGE (Kulhankova et al., 2006), respectively.

Molecular detection has yielded interesting results: (1) Ascomycota are more common in wood than found from fruit body inventories and from direct isolations (Allmer et al., 2006). (2) Species with prolific conidial stages are less common than what would be deduced from isolation work. (3) A wide range of species are detected that previously were not known from the habitat. For example, species that are normally known to be associated with wood of broad-leaved trees have been found in the decay of conifers (Lygis et al., 2004; Vasiliauskas et al., 2004). (4) New functional roles have been implicated for decay fungi. Interestingly, by using DNA-based identification, on several occasions well-known wood-decay organisms (e.g. Phlebia centrifuga, Phlebiopsis gigantea)

have been found in mycorrhizal roots and their ability to develop mycorrhizal root tips under axenic conditions has been confirmed (Vasiliauskas et al., 2007).

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