Location: Horticultural Crops Disease and Pest Management Research Unit
Project Number: 2072-22000-046-048-I
Project Type: Interagency Reimbursable Agreement
Start Date: May 1, 2024
End Date: Sep 30, 2025
Objective:
1) Compare graft indexing of Fragaria to RT-PCR, and high throughput sequencing detection (HTS) methods for viral diseases
2) Compare graft indexing of Rubus to RT-PCR and HTS methods for viral diseases
3) Optimize and update virus detection protocols and provide recommendations to regulatory agencies on disease testing and quarantine time for Fragaria and Rubus, especially on the usefulness of graft indexing.
Approach:
Plants with well-characterized virus profiles will be obtained from the National Clonal Germplasm Repository and the Corvallis-National Clean Plant Network of the U.S. Department of Agriculture Agricultural Research Service (USDA-ARS) in Corvallis, Oregon. These plants will consist of 75 Fragaria and 75 Rubus spp. In addition, G1 plants from the NCPN collection will be used as negative controls.
Objective 1, Known virus infected plants with single infection or mixed virus infections will be grafted onto each strawberry bioindicator lines UC 4, 5, 10 and 11 developed by the University of California strawberry breeding program. The Fragaria bioindicators will be initiated from virus-free tissue culture plants provided Foundation Plant Services (FPS) at UC Davis. After grafting, symptoms will be monitored and recorded weekly until dormancy. The same 75 virus-infected plants will be tested by RT-PCR and subjected to high throughput sequencing (HTS) for known viruses. Tissue samples will be collected at 4 times over 2 years (Villamor, et al., 2022). At each sampling time, stem and leaf and petiole will be collected from each accession and nucleic acids extracted using the Kingfisher Flex and kit according manufactures protocol.
Objective 2, Seventy five known virus-infected Rubus plants (single and mixed infections) will be grafted onto virus free Rubus occidentalis ‘Munger’ plants derived from meristem tissue culture. Symptoms will be recorded weekly until natural dormancy. These 75 Rubus plants will be subjected to RT-PCR tests and HTS. For all the above samples, RNA will be extracted from fresh plant material using the King Fisher automated extraction system to obtain consistent quality. All RT-PCR tests will be performed at the USDA virology lab and the HTS by the FPS, UC Davis. Nucleic acid extraction will be conducted as above.
Objective 3. Results will be analyzed using in-house bioinformatics tools. In cases of disparities between RT-PCR and HTS data plants will be re-sampled to confirm results. If disagreement among test continues then new or improved protocols will be developed and validated. Results will be compared to grafting experiments and conclusions will be forwarded to regulatory agencies for consideration.
