Ectomycorrhizal (ECM) fungi form a mutualistic symbiosis with tree roots and play key roles in forest ecosystems. In return for receiving nutrients and water from the soil via the roots, they receive carbohydrates as photosynthate from their host plants. As is the case for other soil fungal species, the composition of the ECM community is affected by both biotic and abiotic factors; these include climate changes, seasons, soil micro-site heterogeneity, soil and litter quality, host tree species and forest management. To describe in more detail the impact of environmental factors on community composition, long-term, year-round monitoring and a detailed spatial description of the community has to be carried out. However, analyses are very often hindered by a limited sample number and by the ephemeral or cryptic lifestyle of the fungi.
Over the last fifteen years, PCR-based molecular methods and DNA sequencing of nuclear and mitochondrial ribosomal DNA have been used routinely to identify mycorrhizal fungi. However, these methods are timeconsuming and are limited in the number of samples that can be treated in a realistic time frame. With automated molecular genotyping techniques, appropriate DNA databases and a better knowledge of internal transcribed spacer (ITS) variability within fungal species, identification of fungal taxa in environmental samples can now be expanded from the aforementioned methods to high-throughput molecular diagnostic tools, such as phylochips. So far, DNA arrays have been mainly used for genome-wide transcription profiling, but also for the identification of bacterial species from complex environmental samples or for the identification of a few genera of pathogenic fungi and Oomycetes.
Phylochips may comprise up to several thousand probes that target phylogenetic marker genes, such as 16S rRNA in bacteria or the ITS region in fungi. Phylochips have several advantages over traditional approaches, including higher specificity, cost efficiency, rapid identification and detection of target organisms, and the high numbers of samples throughput; therefore, they are increasingly used for the detection of bacterial and pathogenic fungi. In the ECM fungal ecology field, the first application of ribosomal DNA arrays was to develop a specific phylochip (on nylon membranes) to detect Suilloid fungi. Recently, this approach has also been used for truffle identification. No previous study has reported the construction and application of an ECM fungal phylochip to detect a large number of ECM fungal species that belong to various genera from environmental samples. This paper reports the first application of a custom ribosomal ITS phylochip to describe the community composition of ECM fungi on roots. The phylochip carried specific oligonucleotides for 95 fungal species that belong to 25 ECM fungal genera. The specificity of the oligonucleotides was evaluated using ITS amplicons of known reference species. The method was then used to describe ECM fungal communities that were obtained from 30-year-old spruce and beech plantations. The phylochip approach should be an attractive method for routine, accurate and reproducible monitoring of fungal species on specific sites, in which a high sample throughput is required.
Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities. BMC Microbiology 2009, 9: 241 doi:10.1186/1471-2180-9-241
In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were applied. Evaluation of the phylochip by hybridising environmental samples confirmed the possible application of this technology for detecting and monitoring ectomycorrhizal fungi at specific sites in a routine and reproducible manner.
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