Profiling the eukaryotic soil microbiome with differential primers and an antifungal peptide nucleic acid probe (PNA): Implications for diversity assessment

The analysis of 18S rRNA gene amplicons is an important tool to characterize the diversity of the eukaryotic soil microbiome. Here we analyzed two primer sets (TAReuk, EKeuk) and the impact of a newly designed antifungal peptide nucleic acid to enhance the detection of protists in silico and with soil DNA extracted from croplands and a forest. The in silico analyses showed for TAReuk pronounced specificities for protist SAR supergroup and Metazoa, while EKeuk was particularly specific for Ascomycota and Basidiomycota. In silico, the PNA matched with the majority of Ascomycota (81.3 %) and Basidiomycota (65.4 %), but with <6 % of protists. The contrasting primer specificities were confirmed with soil DNA, but the proportion of protist amplicons was similar. In contrast to in silico, effects of the PNA were not as clear with soil DNA, even though it completely inhibited the amplification of the targeted fungal sequences. PNA effects were more pronounced with TAReuk, and results with cropland and forest soil DNA were not consistent, e.g., for cropland, PNA decreased the relative abundance of fungi but for forest it was the opposite, possibly because of different fungal diversity. The divergence between PNA in silico-predictions and results with soil DNA are likely an outcome of primer binding to <100 % complementary target sequences and a still limited DNA sequence databases for soil microbial eukaryotes. With TAReuk, the presence of PNA enhanced the detection of Conosa and, thus, could be a useful tool to study this group in the future.