Submitted to: Mycological Research
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/17/2008
Publication Date: 4/6/2009
Citation: Izzo, A., Mazzola, M. 2009. Hybridization of an ITS-based macroarray with ITS community probes for characterization of complex communities of fungi and fungal-like protists. Mycological Research. 113:802-812. Interpretive Summary: The interactions of fungi that lead to the development of a healthy or diseased plant are very complex. Fungi can impact the plant directly, such as the case with plant pathogens or mycorrhizal fungi. Plant pathogens are constantly challenging the plant and, as in the case of Apple Replant Disease, multiple species can be responsible for a diseased plant. Through a number of interactions with each other and plant pathogens, many fungi can impact plant health indirectly as well. Many taxa exhibit antagonistic behaviour towards other fungi and can keep pathogens in check through mycoparasitism or secretion of caustic compounds. Other taxa compete with pathogens for limiting resources in the soil thereby keeping the inoculum load low in soils. In order to better account for the wide range of fungi impacting plant health, the goal of this study was to develop a tool that had the capacity to simultaneously identify multiple fungi from an environmental sample such as plant tissue or soil. A molecular-based approach was tested, which consisted of a membrane array spotted with the DNA from known species of fungi and was then probed with DNA from complex samples consisting of multiple fungi. Based on our results, this approach can be used to rapidly identify sub-generic lineages of fungi that are present in a sample. Utilization of this approach will improve our ability to account for the many roles that fungi can play in an orchard system and provide a starting point for picking apart more complicated interactions.
Technical Abstract: The ability to characterize fungal community structure and dynamics 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 implementation of this approach generally requires substantial time and financial resources. In this study we evaluated the potential utility of a reverse dot blot approach utilizing macroarrays and probes both consisting of PCR product of the entire fungal ITS1/5.8s/ITS2 region. Such a tool should be more easily attainable method for fungal community profiling and broad diagnostics including both cultivatable and non-cultivatable fungi representing a range of functions (ie. pathogens, saprophytes, mycoparasites). Tests performed using single-species probes within the genus Pythium showed that subgeneric taxonomic lineages could be distinguished when ITS DNA sequence similarity differed by greater than 5-10%. Tests utilizing artificially constructed community probes of known composition successfully detected 8 of the 10 lineages with only one clear false positive. The approach was also successful in identifying prominent taxa in an environmental sample yielding relative signals similar to that observed in other PCR-based analyses (T-RFLP, cloning) of the same environmental sample. A minority of lineages had DNA targets with low melting temperatures and that were not detected on the arrays except under conditions that compromised the specificity of the majority of lineages. Despite this limitation, membrane-based ITS macroarrays coupled with community probes possessed sufficient power to detect multiple subgeneric lineages of fungi in complex samples and should have broad applications in the study of fungal communities.