Hidden Fungi: Combining Culture-Dependent and -Independent DNA Barcoding Reveals Inter-Plant Variation in Species Richness of Endophytic Root Fungi in Elymus repens

The root endophyte community of the grass species Elymus repens was investigated using both a culture-dependent approach and a direct amplicon sequencing method across five sites and from individual plants. There was much heterogeneity across the five sites and among individual plants. Focusing on one site, 349 OTUs were identified by direct amplicon sequencing but only 66 OTUs were cultured. The two approaches shared ten OTUs and the majority of cultured endophytes do not overlap with the amplicon dataset. Media influenced the cultured species richness and without the inclusion of 2% MEA and full-strength MEA, approximately half of the unique OTUs would not have been isolated using only PDA. Combining both culture-dependent and -independent methods for the most accurate determination of root fungal species richness is therefore recommended. High inter-plant variation in fungal species richness was demonstrated, which highlights the need to rethink the scale at which we describe endophyte communities.