Understanding Metabarcoding Benefits and Limitations for Analysis of Phytophthora Communities Neelam Redekar, Joyce Eberhart, Ebba Peterson, and Jennifer Parke, Dept. of Crop and Soil Science, Oregon State University, Corvallis, OR; [email protected] The metabarcoding approach has revolutionized the study of microbial ecology with its ability to detect microbial DNA at an unprecedented depth and coverage. This method exploits high-throughput sequencing and DNA barcoding for identification of microbial species, including novel and uncultivable microbes. The metabarcoding approach is gaining more interest in the Phytophthora scientific community for monitoring the existence and spread of Phytophthora species in diverse habitats. With several case studies from our research in California and Oregon, we will demonstrate benefits of metabarcoding for Phytophthora exploration. We have employed this approach to study Phytophthora diversity in soils from restoration sites, commercial tree seedling nurseries, and organic vegetable farms, and from nursery irrigation water and streams. We investigated some of the operational biases associated with ITS1- amplicon metabarcoding studies using synthetic mock communities and determined the limit of detection for Phytophthora ramorum DNA. In some studies, we compared the metabarcoding approach with a culture-based approach (baiting) for detecting Phytophthora species. We will also present limitations of the metabarcoding approach and highlight challenges we encountered. These include the existence of unresolvable taxonomic groups due to relatively short Illumina sequence lengths, detection of unculturable species, interference of cell-free (relic or environmental) DNA, PCR primer bias resulting in amplification of only a sub-population with matching priming sites, determination of the true biological threshold versus arbitrary cutoffs, and false positives due to barcode-index switching. Understanding and mitigating metabarcoding limitations is crucial for achieving better resolution and accurate determination of the diversity of Phytophthora species. Despite the challenges, the metabarcoding approach continues to be a promising tool for studying Phytophthora ecology. .