Detection of active soil fungi by RT-PCR amplification of precursor rRNA molecules.

Microbial ecologists have used direct RT-PCR amplification of 16S rRNA molecules for the detection of active species of bacteria and archaea, and 18S rRNA molecules for the detection of active fungi. The drawback to this approach for fungi is that 18S rRNA sequences often do not provide sufficient taxonomic resolution to allow identification of taxa in mixed communities to genus or species level. Internal transcribed spacer (ITS) sequences are known to be more taxonomically informative than 18S rRNA sequences and are the common target in DNA based studies but are thought to be absent from RNA pools as they are cleaved after transcription of the large rRNA precursor molecule to leave the mature rRNA's for ribosome synthesis. Here we show, however, that fungal ITS regions can be detected in RNA pools by RT-PCR amplification of fungal precursor rRNA molecules. This suggests that precursor rRNA molecules reside in the cells of active fungi for sufficient time to allow RT-PCR amplification of ITS regions prior to their removal by post-transcriptional cleavage. The RT-PCR conditions for this approach were initially optimised using a range of fungi grown in pure culture prior to applying the approach to complex fungal communities in two contrasting soil types.

Basidiomycete fungal communities in Australian sclerophyll forest soil are altered by repeated prescribed burning.

Soil basidiomycetes play key roles in forest nutrient and carbon cycling processes, yet the diversity and structure of below ground basidiomycete communities remain poorly understood. Prescribed burning is a commonly used forest management practice and there is evidence that single fire events can have an impact on soil fungal communities but little is known about the effects of repeated prescribed burning. We have used internal transcribed spacer (ITS) terminal restriction fragment length polymorphism (T-RFLP) analysis to investigate the impacts of repeated prescribed burning every two or four years over a period of 30 years on soil basidiomycete communities in an Australian wet sclerophyll forest. Detrended correspondence analysis of ITS T-RFLP profiles separated basidiomycete communities in unburned control plots from those in burned plots, with those burned every two years being the most different from controls. Burning had no effect on basidiomycete species richness, thus these differences appear to be due to changes in community structure. Basidiomycete communities in the unburned control plots were vertically stratified in the upper 20 cm of soil, but no evidence was found for stratification in the burned plots, suggesting that repeated prescribed burning results in more uniform basidiomycete communities. Overall, the results demonstrate that repeated prescribed burning alters soil basidiomycete communities, with the effect being greater with more frequent burning.

Diversity of fungi in hair roots of Ericaceae varies along a vegetation gradient.

Ericaceous dwarf shrubs including Calluna vulgaris and Vaccinium spp. occur both in open heathland communities and in forest ecosystems as understory vegetation. Ericaceous shrubs were once thought to form ericoid mycorrhizal associations with a relatively narrow range of ascomycetous fungi closely related to, and including, Rhizoscyphus ericae. However, perceptions have recently changed since the realization that a broader range of ascomycete fungi, and in some cases basidiomycete fungi, can also form associations with the roots of ericaceous plants. We used a combination of molecular approaches, including denaturing gradient gel electrophoresis, terminal restriction fragment length polymorphism, cloning and sequencing, to investigate the diversity of fungi associated with C. vulgaris roots collected across a heathland/native Scots pine forest vegetation gradient. We also determined differences in fungal community composition between roots of co-occurring C. vulgaris and Vaccinium myrtillus in the forest understory. Collectively, the data show that a large diversity of potentially ericoid mycorrhizal fungal taxa associate with roots of C. vulgaris and V. myrtillus, and that ascomycetes were about 2.5 times more frequent than basidiomycetes. The assemblages of fungi associated with C. vulgaris and V. myrtillus were different. In addition, the community of fungi associated with C. vulgaris hair roots was different for samples collected from the forest, open heathland and a transition zone between the two. This separation was partly, but not entirely, due to the occurrence of typical ectomycorrhizal basidiomycetes associated with the hair roots of C. vulgaris in the forest understory. These data demonstrate that forest understory ericaceous shrubs associate with a diverse range of ascomycete and basidiomycete taxa, including typical ectomycorrhizal basidiomycetes.