Needle in a Haystack: Culturing Plant-Beneficial Helotiales Lineages From Plant Roots

Root-associated Helotiales fungi are increasingly recognised as beneficial fungal partners promoting plant growth under nutrient-limited conditions, particularly, in non-mycorrhizal hosts lacking the ancestral arbuscular mycorrhizal symbiosis. However, the ecology of these fungi is still cryptic as relatively few lineages have been successfully cultivated from roots for further study. Here, we attempted the mass isolation of root endophytic fungi to evaluate the recovery of known plant-beneficial Helotiales lineages using a tailored culture-based approach. We sampled six wild non-mycorrhizal species from the Brassicaceae, Caryophyllaceae, and Cyperaceae, growing in nutrient-limited alpine soils. We isolated 602 root endophytes and compared this culturable diversity with the one observed via fungal ITS2 metabarcoding. Metabarcoding revealed that Helotiales taxa dominated the fungal communities, with 43% of these detected taxa also represented in our collection. Accordingly, most root endophytes in our collection (53%) were Helotiales. These isolates, some with P solubilisation potential, belonged primarily to three Helotialean clades and were phylogenetically related to plant growth-promoting or mycorrhizal-like strains. This analysis highlights that the roots of alpine non-mycorrhizal plants harbour diverse plant-beneficial root-endophytic Helotiales, and the isolates obtained are a promising resource to explore the plant-beneficial mechanisms and ecological traits of these fungi.