Development of an amplicon-metagenomic approach based on MiSeq Illumina sequencing for the study of Phytophthora spp. diversity in water samples

"An amplicon-metagenomic approach was used to investigate Phytophthora spp. diversity in waters collected from four Scottish natural habitats, i.e. Invergowrie, Glensaugh Research Station, Cairngorms National Park and Sourhope Research Station. Sequencing of the rDNA ITS1 barcode-region on an Illumina MiSeq platform with genus-specific primers produced around 700,000 sequences that were clustered into 4,671 OTUs using a 99% similarity threshold. Assigning taxonomy to the representative sequences of the OTUs was done using the BLAST algorithm and a manually curated Oomycetesdatabase. The results showed that majority of the sequences were associated to the genus Phytophthora. Although few OTUs were associated to other Oomycetes, the use of genus-specific ITS1 primers allowed understanding well the diversity of Phytophthora population. Moreover, the results revealed a clear site-to-site variation, where the lowest species diversity was found in GSB Glensaugh and the highest diversity at Invergowrie. The analysis of ?-diversity, based on Bray Curtis matrices, confirmed differences (P<0.001) in populations associated to the investigated environments, with only few OTUs shared among samples. This approach proved effective in the study of Phytophthora spp. diversity and the use of genus-specific primers contributed to an unprecedented level of accuracy, compared to traditional techniques based on direct isolation, while preserving sensitivity of identification and relative quantification of Phytophthora species. This study demonstrated that Illumina sequencing of the ITS1 region may serve to study in depth diversity and ecological niches of Phytophthora species."