Provided that the corresponding oligonucleotides were included on the array, all species that were detected by cloning-sequencing could also be
identified with the phylochip. As the corresponding oligonucleotides were lacking on the phylochip, species belonging to the Atheliaceae, Sebacinaceae or Pezizales HSP cancer were not detected. Furthermore, the comparison of array signal intensity with ITS sequence frequency in the ITS clone library revealed the potential of the phylochip to detect taxa that were represented by approx. 2% of DNA types in the amplified DNA sample. However, the quantitative potential of this custom phylochip remains to be further accessed as bias linked to the PCR amplification could take place. The phylochip also detected species that were not expected
according to the results obtained from the use of the other two approaches. This could be due to cross-hybridisations and/or to the fact that these under-represented species in the community could not be detected by the other Selonsertib datasheet approaches as the rarefaction curves of the ITS library sequencing method did not reach a plateau (HDAC inhibitor additional file 1). When compared to each other, both of the other approaches provided similar, but not identical, profiles of the ECM communities. Approximately 70% of the species were detected using either method individually (Table 1). For the beech sample, three species were detected only by morphotyping as the PCR amplification of their DNA using ITS1F/ITS4 and/or NSI1/NLB4 primer pairs failed. Tedersoo et al. [35] showed that PCR of ITS from several ECM species failed using these universal fungal rDNA primers, and they stressed the need for additional taxon-specific PCR
primers to be used for comprehensive genotyping of ECM communities. One of the morphotypes detected in the beech sample was a Lactarius species. In the same root sample, a Pezizales species was found by ITS-sequencing and cloning/sequencing; this suggests a possible co-colonisation of the ECM root tip [36]. ECM root tips can be colonised by more Cyclin-dependent kinase 3 than one fungal taxon, by two different ECM species, or by one ECM species and an endophytic or parasitic species. Typically, these species are overlooked by the use of only morphotyping, but they can be detected by molecular biological approaches. Conclusion In this study, we demonstrated that identification of ECM fungi in environmental studies is possible using a custom phylochip. The detection of most of the species by the phylochip was confirmed by two other widely used detection methods. Although the possible application of the phylochip technique to other study areas is dependent on the fungal species to be analysed, high-quality sequence support for several temperate and boreal forest ecosystems is found in databases such as UNITE [11].