Comparative Metabarcoding of ITS1, ITS2, and Full-Length ITS Reveals Marker- and Tissue-Specific Variation in Fungal...

Accurate profiling of plant-associated fungal and oomycete communities depends critically on the choice of ITS marker. Using Illumina sequencing for ITS1/ITS2 and PacBio HiFi sequencing for full-length ITS (ITSf), we compared read recovery, taxonomic assignment depth, and diversity patterns across potato leaf and root tissues. ITS2 generally recovered more diverse and even community profiles in leaves, whereas ITSf recovered comparatively more even and taxonomically broad profiles in roots. In contrast, ITS1 showed high read recovery but produced strongly skewed compositional profiles and was frequently dominated by host-derived sequences. Beta-diversity analyses indicated that ITS marker choice was associated with substantial variation in observed community composition, while functional annotation highlighted communities composed of taxa associated with multiple ecological guilds, including endophytes and opportunistic pathogens such as Cladosporium and Ilyonectria. Overall, the results demonstrate that ITS marker choice strongly influences the observed structure and diversity of plant-associated communities. ITS2 was generally more suitable for phyllosphere-associated communities, whereas ITSf provided broader recovery of root-associated taxa. Combining complementary markers therefore offers a more comprehensive representation of potato-associated microbial eukaryotic communities than single-marker approaches alone.