Strong primer bias for <i>Tulasnellaceae</i> fungi in metabarcoding: Specific primers improve the characterization of the mycorrhizal communities of epiphytic orchids

Abstract

Primer bias toward Tulasnellaceae 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 Tulasnella-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 Ceratobasidiaceae and Serendipitaceae fungi but did not reflect Tulasnellaceae diversity. Tulasnellaceae fungi were mainly detected using the Tulasnella-specific primer pair. These tendencies were observed in both the mock community and environmental samples. These results strongly suggest that the use of a Tulasnella-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.

Orchid mycorrhizal fungi and ascomycetous fungi in epiphytic Vanda falcata roots occupy different niches during growth and development

“…Generally, it is accepted that orchids with high mycorrhizal specificity are most likely to be rare due to high dependency on OMF distribution in their natural habitats. In the case of V. falcata, its distributions were constricted from central to southern Japan with various types of phorophytes (Suetsugu et al 2015;Rammitsu et al 2019Rammitsu et al , 2021. Accordingly, V. falcata has high phenotypic variation even for wild plants across Japan, causing such extensive association to be beneficial to the plant for distribution.…”

“…Previous studies suggested that vandaceous orchids-especially those of subtribe Aeridinae-mainly associated with Ceratobasidium, proven by metabarcoding and seed germination results (Otero et al 2002;Yukawa et al 2009;Hoang et al 2017;Mújica et al 2018;Rammitsu et al 2019;Kendon et al 2020). Although Ceratobasidium strains have also been isolated from V. falcata (Rammitsu et al 2021), mycobionts for seed germination have never been reported. Thus, this study analyzed the whole fungal community in seeds and root samples of V. falcata using amplicon sequencing.…”

Spatial turnover of fungi and partner choice shape mycorrhizal networks in epiphytic orchids

“…Perhaps because of the use of an additional specific primer pair, Tulasnellaceae were the most abundant OMF in nearly all orchid species. However, the need to use specific primers to amplify this family is largely acknowledged and has been recently demonstrated by Rammitsu et al (2021). Tulasnellaceae and Serendipitaceae were both represented by numerous OTUs whereas only three OTUs of Ceratobasidiaceae were detected, only one being frequent, suggesting that Ceratobasidiaceae might be rarer in these epiphytic rainforest habitats (Martos et al, 2012).…”

Spatiotemporal dynamics and functional characteristics of the composition of the main fungal taxa in the root microhabitat of Calanthe sieboldii (Orchidaceae)

“…However, the experimental approach of using two primers may have distorted these comparative results regarding the three spatial classes of fungal communities. Different primers have preferences for various taxa, and this may introduce systematic bias into the results [68,69]. This limitation implies that results concerning comparisons between plant roots and soil need to be interpreted cautiously.…”