Hybridization of an ITS-based macroarray with ITS community probes for characterization of complex communities of fungi and fungal-like protists.

The ability to characterize fungal community structure and dynamics in the environment is constantly challenged by the high levels of diversity that must be confronted. Large-scale oligonucleotide arrays for use in such analytical studies are currently under development; however, the implementation of this approach generally requires substantial time and financial resources. To address the need for a more accessible tool for fungal community profiling and broad diagnostics, we evaluated the potential utility of a reverse dot blot approach utilizing macroarray targets and probes that each consisted of a PCR product of the entire fungal ITS1-5.8S-ITS2 gene region. Samples used to generate the array targets included both culturable and non-culturable fungi and fungal-like protists representing a range of ecological functions. Tests performed using single-species probes within the genus Pythium demonstrated that taxonomic lineages could generally be distinguished when ITS DNA sequence similarity differed by greater than 5-10%. An artificially constructed community probe of known composition successfully detected eight of the 10 lineages contained on the array with only one clear false positive in 95 targets. The approach was also successfully applied to environmental samples. Taxa resident in the soil of a local orchard were identified using the array and matched those documented in previous studies. Closely related taxa from a previously uncharacterized and geographically distant orchard soil were also identified by the array and had affinities to Leptodontium, Cadophora, Zalerion, and Geomyces. These taxa were further confirmed to be present in the sample by cloning and DNA sequencing. A minority of lineages had DNA targets with low melting temperatures which were not detected on the arrays except under conditions that compromised specificity. Membrane-based ITS macroarrays coupled with community ITS probes possessed sufficient power to detect multiple genus-level lineages of fungi in complex samples and should have broad applications in the study of fungal communities.