Comparison of Molecular and Morphological Identification Methods for Anguina Seed Gall Nematodes in Oregon Grasses Grown for Seed

Abstract

Oregon's grass seed industry specializes in the production of forage grasses, including annual ryegrass ( Lolium multiflorum) and orchardgrass ( Dactylis glomerata). These species are hosts of seed gall nematodes (SGNs): Anguina funesta and Anguina sp. SGNs cause yield-limiting seed galls and can also vector toxic Rathayibacter bacteria. Trade partners have strict phytosanitary regulations leading to rejection of seed lots infested with SGNs. Current best practices for SGN detection focus on postharvest seed evaluation. Methods to evaluate fields before harvest could improve risk management decisions. In this study, we evaluated timing, collection, and detection methods to generate new recommendations for SGN detection throughout the growing season. Fields of annual ryegrass (21) and orchardgrass (7) were sampled in the 2022 and 2023 growing seasons at tillering (March), flowering (May), harvest (July), and germination (November). At each time point, tillers, seed heads, or soil samples were collected. Nematodes were extracted from soil, tiller, and seed head samples using traditional nematology methods. Alternatively, SGN-specific real-time and conventional PCR protocols were evaluated on DNA extracted from tillers or seed heads. Direct enumeration of SGN from tillers with traditional nematology methods resulted in positive detections in 11 to 19% of fields, depending on sample time and year, as opposed to 33 to 44% of fields when using molecular methods. SGNs were detected in 40% of fields using both methods when evaluating seed head samples. This study indicates the utility of incorporating molecular methods for risk evaluations of SGNs and provides recommendations for accurate SGN detection throughout the growing season. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 “No Rights Reserved” license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.