Location: Crops Pathology and Genetics Research
Project Number: 2032-22000-017-005-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2019
End Date: Aug 31, 2024
1)Identify alternative hosts and reservoirs of grapevine red blotch virus (GRBV). 2)Determine presence and biology of potential treehopper vectors in symptomatic vineyards and associated crop and non-crop landscapes. 3)Improve knowledge of GRBV acquisition and transmission by its vector(s). 4) Develop cost-effective GRBV diagnostic tools.
Insect host plants will be tested for GRBV presence by PCR or qPCR. For any non-Vitis host that test positive for GRBV, additional tests will be done to confirm virus replication in the host by total nucleic acid extraction, digestion with DNAse, then RT-PCR to confirm the presence of virus specific RNA in the plant. Conduct in-depth insect vector-related studies with focus on treehoppers (Membracidae) associated with vineyards. Vineyards where virus spread is suspected representing both CA and OR will be visually surveyed for treehoppers and other phloem-feeding Hemiptera using sweep net, beat sheet, sticky trap, or vacuum sampling as appropriate. Characterize GRBV transmission by determining acquisition and inoculation access periods, latent period, virus retention, and persistence of vector infectivity to increase knowledge of transmission. Develop a field deployable method that can be used to detect GRBV by testing laboratories and vineyard personnel. Two methods (1) LAMP methods (Loop-amplification technology) and thermostable helicase dependent amplification (tHDA), currently under development in Sudarshana lab, will be compared for simplicity, sensitivity and cost of deployment. We have already designed a pair of primers for tHDA-mediated method for use in combination with EvaGreen, dsDNA binding dye for real-time monitoring of the amplification process. This will be compared with latest pH indicator-based monitoring of DNA amplification during LAMP. Amplified DNA will also be examined using a field deployable portable blue LED lamp.