Strong Primer Bias for Fungi in Metabarcoding: Specific Primers Improve the Characterization of the Mycorrhizal Communities of Epiphytic Orchids

Primer bias toward fungi during PCR is a known issue with metabarcoding analyses for the assessment of orchid mycorrhizal communities. However, this bias had not been evaluated for the fungal communities of epiphytic orchids, which account for 69% of all orchid species diversity. We compared the mycorrhizal communities detected using two primer pairs, a fungal universal primer pair (ITS86F/ITS4) and -specific primer pair (5.8STulngs/ITS4-Tul2), using a mock community of fungal isolates from epiphytic orchids and also environmental samples, including orchid roots and a tree bark tip from the host tree of an epiphytic orchid collected. The detected mycorrhizal communities differed widely depending on the primer pairs used. The fungal universal primer pair successfully identified and fungi but did not reflect diversity. fungi were mainly detected using the -specific primer pair. These tendencies were observed in both the mock community and environmental samples. These results strongly suggest that the use of a -specific primer in combination with a fungal universal primer is essential for assessing the mycorrhizal communities of orchids through metabarcoding analysis, especially in epiphytic orchids. Our study contributes to further understanding of the diversity of mycorrhizal fungi in orchids.