Comparison of Culturing and Metabarcoding Methods to Describe the Fungal Endophytic Assemblage of Brachypodium rupestre Growing in a Range of Anthropized Disturbance Regimes

SIMPLE SUMMARY: The richness (number of species) of the fungi kingdom is estimated at 1.5 million species, but the vast majority remains unknown. Many of them inhabit plants—the so-called fungal endophytes—and may establish different types of interactions with their host plant. Fungal endophytes have been traditionally studied by letting them grow in appropriate culturing media in petri dishes, but novel massive DNA sequencing techniques which do not require a cultivation step (metabarcoding) are gaining ground. Both techniques were applied and compared to characterize the mycobiome of plants of a tall grass (Brachypodium rupestre) growing in high-mountain grasslands with different plant diversity (low and high). The two methods showed similar trends comparing endophyte richness between plant tissue types (root > rhizome > shoot) and between grasslands (low-diversity > high-diversity). However, the metabarcoding identified almost six times more endophyte species than the culturing although the most isolated fungal species via culturing, Omnidemptus graminis, was not recognized via metabarcoding. We conclude that the complementation of both techniques is still the best option to obtain a complete characterization of the fungal endophytic assemblage of the plant species. ABSTRACT: Fungal endophytes develop inside plants without visible external signs, and they may confer adaptive advantages to their hosts. Culturing methods have been traditionally used to recognize the fungal endophytic assemblage, but novel metabarcoding techniques are being increasingly applied. This study aims to characterize the fungal endophytic assemblage in shoots, rhizomes and roots of the tall grass Brachypodium rupestre growing in a large area of natural grasslands with a continuum of anthropized disturbance regimes. Seven out of 88 taxa identified via metabarcoding accounted for 81.2% of the reads (Helotiaceae, Lachnum sp. A, Albotricha sp. A, Helotiales A, Agaricales A, Mycena sp. and Mollisiaceae C), revealing a small group of abundant endophytes and a large group of rare species. Although both methods detected the same trends in richness and fungal diversity among the tissues (root > rhizome > shoot) and grasslands (low-diversity > high-diversity grasslands), the metabarcoding tool identified 5.8 times more taxa than the traditional culturing method (15 taxa) but, surprisingly, failed to sequence the most isolated endophyte on plates, Omnidemptus graminis. Since both methods are still subject to important constraints, both are required to obtain a complete characterization of the fungal endophytic assemblage of the plant species.