DNA and RNA metabarcoding reveal distinct seed-borne mycobiota

Abstract Background Tree seeds harbor diverse fungal communities, including both pathogens and mutualists, that can influence plant health. These communities comprise living, metabolically active organisms as well as dormant or dead cells. Because only active fungi interact with their hosts, distinguishing active from inactive taxa is crucial, especially for environmental and phytosanitary monitoring. Traditional culturing methods capture living fungi but account for only a small fraction of the total fungal diversity. Currently, these methods are increasingly replaced by high-throughput DNA metabarcoding, which detects a broader range of taxa. However, DNA persists after cell death and occurs in dormant cells, preventing distinction between active and inactive fungi. In contrast, RNA metabarcoding detects metabolically active organisms and may better reflect living fungal communities than the other two methods, though its use in assessing plant-associated fungi remains underexplored. We used culturing, DNA-, and RNA-based metabarcoding to compare fungal communities associated with seeds of three key European tree species ( Fagus sylvatica , Abies alba , Pinus sylvestris ). Results DNA and RNA metabarcoding detected largely distinct, non-overlapping fungal communities, with differences primarily driven by rare active taxa in the RNA dataset. Several cultured genera—likely representing abundant and metabolically active taxa—were shared between both metabarcoding approaches. Conclusions These results highlight the complementary nature of the three methods for characterising seed-associated fungi. Combining culturing, DNA- and RNA-based metabarcoding may provide the most comprehensive assessment of fungal diversity, while RNA metabarcoding alone offers a promising opportunity to identify the active members of fungal communities for improved environmental and phytosanitary monitoring.