Location: Forage Seed and Cereal Research Unit
Project Number: 2072-12620-001-023-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Mar 1, 2022
End Date: May 31, 2023
Objective:
Anguina funesta and other Anguina spp. seed gall nematodes can vector Rathayibacter toxicus bacteria to forage grasses including annual ryegrass, perennial ryegrass, and orchardgrass. Anguina spp. can lead to exported seed lots to be rejected in other countries and R. toxicus causes Ryegrass Toxicity (RGT) that can kill grazing animals. Currently there is a need for accurate and early identification of the nematode vector of this pathogen system to support growers shipping abroad and to be ahead of any RGT outbreaks. The purpose of this work is to improve survey and identification methods for the nematode vectors in this disease system, in order to understand current vector prevalence, quickly respond to future outbreaks, and reduce the risk of introducing the Anguina – R. toxicus disease complex to the grass seed industry of the United States. To meet this goal, we will utilize the following objectives: 1) Evaluate survey timing (spring or fall) and collection (soil, seed, tillers) for the most accurate detection of A. funesta and other Anguina spp. associated with annual ryegrass, perennial ryegrass and orchardgrass grown for seed in Oregon; and 2) develop a video-based nematode identification tutorial to build capacity among other diagnosticians/laboratories.
Approach:
To develop better survey tools, we will collect soil, plant, and seed samples throughout the Willamette Valley of Oregon; five each of annual ryegrass, perennial ryegrass and orchardgrass (15 fields total). We will survey for Anguina spp. at two time points: in the fall (October, after planting) and spring (March), before any seed set has occurred. At each sampling, six 100-foot transects will be walked to collect soil and plants at every 10 feet (ten 6” soil cores and ten plant tiller samples per transect). Soil and tiller samples will be bulked by transect (6 soil and 6 tiller samples per field) before nematode extraction. Nematodes will be extracted from soil using decanting and sieving followed by density centrifugation. Extraction of nematodes from plant material will be conducted under intermittent mist. Extracted nematodes will be morphologically identified and characterized with a microscope. A third field visit, after seed set but before harvest (May), will be conducted to walk the same transects and rate incidence of any visible seed galling. If galls are detected, they will be collected, bulked by transect and evaluated under intermittent mist for nematode recovery and identified morphologically as described above.
For molecular identification we will extract DNA directly from root material and use a quantitative Polymerase Chain Reaction (qPCR) protocol to discern four agriculturally important Anguina spp. (A. agrostis, A. funesta, A. pacificae, and A. tritici) from a diagnostic-like sample. We will compare these results to our morphological identifications to help verify the utility of the assay in a diagnostic setting. We will select one root sample (~100 mg of tissue) per transect per sampling time (180 total) for this molecular analysis.
We will compare soil and plant recovery of nematodes, and seed gall disease incidence from the fall and spring sampling times. Along with our molecular identification, we will develop a standardized protocol for Anguina sampling in Oregon grass for seed fields. If pure cultures of any Anguina species can be obtained, especially A. funesta, and if symptoms resembling Rathayibacter toxicus infection are detected, we will attempt to recover the pathogens in pure culture for future greenhouse studies.
Multiple Anguina spp. are associated with grasses grown for forage production. Their differentiation morphologically is difficult; therefore, a webinar-based identification course will be developed to share with laboratory diagnosticians tasked with identifying these nematodes. The webinar will also provide recommendations on qPCR methodologies available for Anguina identification. With the support of the American Phytopathological Society (APS), a webinar will be developed to provide identification training for Anguina species associated with grass. In general, audiences of 40-100 participants can be expected for APS webinars.
