Fungal Succession And Interactions In Forest Litter A Molecular Approach

One of the major challenges in studying fungal ecology is identification of the fungi present in a particular substrate. Traditionally, litter fungi have been studied primarily through isolation of fungi growing out from litter components placed on artificial media (Frankland et al., 1990). During the last 15 years, molecular methods have been developed for fungal identification and have been used extensively to analyse communities of mycorrhizal fungi (Horton and Bruns, 2001). Only recently have techniques been developed that enable DNA-based identification of mixed fungal communities directly from natural substrata (Landeweert et al., 2003), and the first molecular studies of fungal communities in forest litter have been published (O'Brien et al., 2005; Lindahl et al., 2007). The major advantage of DNA-based methods over identification of isolated fungi is that analysis of extracted DNA does not discriminate against non-culturable and slow-growing taxa. Molecular methods may, in fact, provide very different views of fungal communities than isolation-based methods (Allmer et al., 2006). In addition, sequence comparisons with databases may enable identification of non-sporulating taxa, including basidiomycetes. The major drawbacks of molecular identification techniques are the large costs involved and the required access to a molecular biology laboratory. In addition, molecular techniques suffer from the same problems as most other methods, when attempting to analyse complex microbial communities from limited numbers of samples (Taylor, 2002).

Briefly, the methods are based on polymerase chain reaction (PCR) amplification of a small part of the fungal genome (usually the ITS region) using fungal-specific primers (White et al., 1990) and DNA extracted from field samples as template. The PCR products, containing a mixture of DNA from many different fungal taxa, are incorporated into plasmid vectors and cloned into bacteria. When the transformed bacteria are cultured on agar plates, each bacterial colony originates from a single bacterial cell and thus contains DNA from a single fungal taxon. Bacterial clones (e.g. 25-250 per sample) are collected and the cloned fungal DNA is re-amplified and sequenced (Landeweert et al., 2003). Taxonomic identities may be assigned by comparisons with reference sequences obtained from identified sporocarps or sporulating cultures. Cloning is costly and involves a large amount of laboratory work, and if large numbers of samples are analysed, the cloning approach has to be combined with a community fingerprinting method, such as terminal restriction fragment length polymorphism (TRFLP). We have conducted a series of studies where DNA was extracted from needle litter, moss litter, humus or soil from Swedish coniferous forests, and the total fungal community was analysed by cloning, sequencing and TRFLP analysis. Some of the results are outlined below (Lindahl et al., 2007 and unpublished data).

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