Comparison of primers for the detection of Phytophthora

Many oomycetes are important plant pathogens that cause devastating diseases in agricultural fields, orchards, urban areas, and natural ecosystems. Limitations and difficulties associated with isolating these pathogens have led to strong uptake of DNA metabarcoding and mass parallel sequencing. At least 20 primers combinations have been designed to amplify oomycetes or, more specifically, Phytophthora species from environmental samples. We used the Illumina sequencing platform to compare 13 primer combinations on mock communities and environmental samples. The primer combinations tested significantly varied in their ability to amplify Phytophthora species in a mock community and from environmental samples; this was due to either low sensitivity (unable to detect species present in low concentrations) or a lack of specificity (an inability to amplify some species even if they were present in high concentrations). Primers designed for oomycetes underestimated the Phytophthora community compared to Phytophthora-specific primers. We recommend using technical replicates, primer combinations, internal controls, and a phylogenetic approach for assigning a species identity to OTUs or ASVs. Particular care must be taken if sampling substrates where hybrid species could be expected. Overall, the choice of primers should depend upon the hypothesis being tested. The data files presented here contain the raw data for four separate metabarcoding runs analysed for this study in a fasta format