Location: Forage Seed and Cereal Research Unit
Project Number: 2072-21600-001-025-I
Project Type: Interagency Reimbursable Agreement
Start Date: Jul 15, 2026
End Date: Jul 14, 2027
Objective:
Obj. 1 – Compare resolution of curated 18S and COX1 databases with data generated from nanopore sequencing of extracted PPN.
Obj. 2 – Validate nanopore sequencing of PPN communities to identify and quantify a single species of concern (Meloidogyne naasi or M. chitwoodi) in turfgrass and potato soils.
Obj. 3 – Validate nanopore sequencing for the detection and quantification of key PPN community members in turfgrass and potato soil samples.
Approach:
Obj. 1. To evaluate the compatibility of these databases with nanopore sequencing results, mock PPN communities will be generated with 15 different PPN likely to be found in Oregon turfgrass samples. The mock communities will be generated by combining individual juvenile and adult PPN by manually picking them from soil samples of turfgrass samples throughout the state. Samples will be collected from January to March of 2026 with the assistance of a contractor, by pulling twenty 10-cm samples per green from up to 20 greens in the state. Nematodes will be extracted from 100 cc of soil by sieving/sugar centrifugation. Recovered nematodes will be evaluated on a compound microscope. Four mock communities will be generated. Nucleic acid extraction will be conducted using Qiagen Power Soil Kits. Following DNA extraction, for each mock community extraction, the 18S or COX1 gene region will be amplified. PCR will be run in triplicate, totaling 6 reactions per community and 24 reactions overall. PCR products will be barcoded using the Oxford Nanopore Technologies Native Barcoding Kit and pooled by gene region for processing on two flow cell sequencing runs (one 18S run, one COX1 run). Nanopore sequencing will be conducted on a MinION Mk1D, and base calling, demultiplexing, and trimming will be conducted using MinKNOW software from Oxford Nanopore. After a consensus sequence is generated for each gene region, operational taxonomic units (OTUs) will be identified using the database(s) for the target gene region, i.e., 18S data will be compared in both NemaTaxa and 18SNemaBase, COX1 data will be evaluated in the Nema-mtDB. Resultant OTUs will be compared to the original mock community counts and across databases/primer sets. Obj. 2. To determine the detection limit of nanopore sequencing for M. naasi or M chitwoodi, mock populations of multiple species of root knot nematode from Oregon will be collected from established cultures grown on wheat or tomato plants. Eggs will be extracted from roots using a bleach extraction method, and then placed on hatching chambers for 3-7 days to hatch J2s for experiments. Mock Meloidogyne spp. communities will be created to determine the limit of detection at the individual scale from other closely related species. Three independent replicates of each community will be generated (n = 24). Mock communities will be processed as in Obj. 1. Taxonomic differentiation of Meloidogyne species from sequencing results will be compared. Obj. 3: Samples will be collected from Oregon research farms specializing in turfgrass and potato. Between January and March 2026, 5 turfgrass plots, and 5 potato plots will be sampled that have a known history of Meloidogyne spp. presence. Recovered PPN will be concentrated in 10 mL and will be microscopically identified and quantified. The same sample will then be processed as described in Objective 1 for both 18S and COX1 PCR and barcoding. Nanopore sequencing will be conducted in 2 runs and analyzed. Results will be compared to action thresholds for both turfgrasses and potatoes.