Full-Length ITS Amplicon Sequencing Resolves Phytophthora Species in Surface Waters Used for Orchard Irrigation in California's San Joaquin Valley

Diverse Phytophthora species, including many important plant pathogens, have been widely detected among surface water irrigation sources. In the past decade, metabarcoding has been used to characterize waterborne Phytophthora populations. Metabarcoding typically involves amplification of portions of the nuclear ribosomal internal transcribed spacers (ITSs) ITS1 or ITS2 from Phytophthora species, followed by indexed high-throughput sequencing. However, full-length sequences of the entire ITS region are required for resolution of many Phytophthora species. We used metabarcoding with Pacific Biosciences (PacBio) sequencing of full-length ITS amplicons to analyze populations of Phytophthora in waterways of the Stockton East Water District (SEWD) in the northern San Joaquin Valley of California. This approach yielded species-level resolution of many members of the Phytophthora community. Results were compared with those obtained by using ITS1 or ITS2 regions alone and were found to provide superior species resolution for P. pini, P. capsici, and P. gregata. Samples were collected throughout the 2021 irrigation season from five waterways across the SEWD. Thirty-eight Phytophthora species were detected in the waterways, including tree-crop pathogens P. acerina, P. cactorum, P. pini, P. x cambivora, P. niederhauserii, P. mediterranea, and P. taxon walnut. These pathogenic species were detected throughout the SEWD during most of the irrigation season. The results demonstrated the usefulness of full-length ITS amplicon sequencing for identifying Phytophthora species in environmental samples and suggested that some disease risk may be incurred by orchardists irrigating with SEWD water. Additional epidemiological studies will be required to critically evaluate this risk.