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Phytophthora  is one of the most important and aggressive plant pathogenic genera in agriculture and forestry. Early detection and identification of its pathways of infection and spread are of high importance to minimize the threat they pose to natural ecosystems. eDNA was extracted from soil and water from forests and plantations in the north of Spain.  Phytophthora -specific primers were adapted for use in high-throughput Sequencing (HTS). Primers were tested in a control reaction containing eight  Phytophthora  species and applied to water and soil eDNA samples from northern Spain. Different score coverage threshold values were tested for optimal  Phytophthora  species separation in a custom-curated database and in the control reaction. Clustering at 99% was the optimal criteria to separate most of the  Phytophthora  species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct  Phytophthora  species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low  Phytophthora  diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams showed no close match to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess  Phytophthora  species diversity in natural ecosystems.

The Use of Genus-Specific Amplicon Pyrosequencing to Assess Phytophthora Species Diversity Using eDNA from Soil and Water in Northern Spain