Assessing Soil Pathogens via Ribosomal Markers

Monitoring microbial diversity in agricultural soils is crucial for assessing ecosystem health and managing soilborne diseases. Environmental DNA metabarcoding offers a rapid and cost-effective approach for detecting protist pathogens, yet the performance of different ribosomal markers remains insufficiently evaluated. Here, we compared three ribosomal markers of increasing length, a short fragment (V4 region of the 18S rRNA gene, ~450 bp), a medium fragment (18S, ~1,800 bp), and a long fragment (ribosomal operon: 18S–ITS–28S, ~4,500 bp) to assess their effectiveness in detecting protist pathogens in soils from two agricultural sites, one infested with Plasmodiophora brassicae. All markers and the bioinformatic pipeline were validated using DNA from reference cultures. The medium 18S fragment yielded the highest proportion of protist reads, particularly for Stramenopiles (oomycetes), but missed other major clades. The short V4 fragment detected a broader range of pathogenic lineages, including P. brassicae, but lacked resolution for species-level assignments within Pythium. The long ribosomal operon fragment produced fewer reads but improved phylogenetic resolution. These findings confirm the complementary strengths and limitations of each marker: shorter fragments maximize detection breadth, while longer fragments enhance taxonomic resolution. This study provides a methodological framework for optimizing eDNA-based monitoring of protist pathogens in agroecosystems.