From extensive clone libraries to comprehensive DNA arrays for the efficient and simultaneous detection and identification of orchid mycorrhizal fungi

A DNA array was developed from extensive clone library sequence data sets for the assessment of dominant members of mycorrhizal fungi that associate with terrestrial orchid species. As a-proof-of-concept, the array was developed for the basidiomycetous mycorrhizal partners from three closely related perennial Orchis species, including Orchis anthropophora, O. militaris and O. purpurea. Based on internal transcribed spacer regions, oligonucleotides were developed for seven operational taxonomic units (OTUs; defined as groups of sequences sharing at least 97% sequence similarity), corresponding to members of the Tulasnellaceae family. In order to cover a broader spectrum of tulasnelloid fungi, oligonucleotides were as well developed for two subsets of closely related OTUs. The array was evaluated using multiple primer pairs. In addition, hybridization results were validated by recovery and sequencing of the hybridized amplicons as well as by hybridizing reference DNA samples. Considering the unlimited expansion possibilities of DNA arrays to include specific detector oligonucleotides for other and more microorganisms, the method described here has the major advantage that it provides a powerful, rapid and cost-effective way for the simultaneous detection and identification of a wide range of orchid mycorrhizae. The design, development and advantages of the array are discussed in relation to its potential for future research in mycorrhizal ecology.