Detection of Wood Destroying Fungi Using DNA Microarray Technology
K Jacobs, N Rangno, W Scheiding, B Weiss, D Müller, C Hiller, W Brabetz
Wood decay fungi of the phylum Basidiomycota cause serious damage to wooden constructions and building elements. The elimination and the appraisement of fungal decay require an assured species identification. Conventional fungal diagnostics are mainly based on morphological characteristics by macro- and microscopy analysis. For some years, standardised and highly sensitive molecular methods focussing on DNA analysis are increasingly used. However, these methods still have some disadvantages in routine diagnostics, like long operating time and infeasibility of analysing mixed samples, where several fungi occur simultaneously in one sample. To overcome these drawbacks, a DNA microarray for the confident identification of the 27 most important wood destroying basidiomycetes was developed that is already used in the laboratory routine. First of all, species-specific DNA probes were generated on the basis of validated rDNA ITS sequences from 121 fungal isolates. The specificity of these DNA probes was assured using the polymerase chain reaction (PCR) and microarray detection technology. Afterwards, the microarray detection procedure that is based on the arrayed ligation reaction (ALR) was optimized and validated. For a comprehensive field testing and validation of the newly developed detection technique, 36 samples from decayed timber have been collected. The test specimens were analysed by microarray analysis as well as by conventional methods like microscopy analysis and ITS sequencing determination in parallel. Thereby, at 33 samples the fungus species could be addressed directly by microarray analysis. In the case of three samples, the concentration and the quality of the DNA preparations were insufficient for a distinct diagnosis. In these samples the conventional morphological identification as well as the sequencing determination failed, too. The comparison methods provided a higher level of failure, in which 22 % (ITS sequencing), respectively 33 % (conventional diagnostics) of the samples could not be identified to the species level. The field test showed that the microarray based diagnostic system ensures a fast and reliable identification of wood decay fungi. Since January 2010, the Mycotype® BasidioQS Microarray Detection Kit is available as the first commercial product. This DNA microarray was developed in a research cooperation between the Institute of Wood Technology, Dresden (IHD) and the Biotype Diagnostic GmbH, Dresden.
Keywords: wood decay fungi, fungal diagnostics, DNA microarray