2013 Annual Report
1a.Objectives (from AD-416):
1. Develop and validate diagnostic tests for the viruses involved in these complexes and transfer these validated tests to interested parties; 2. Identify candidate virus vectors based on virus genomics with greenhouse transmission testing; 3. Identify virus combinations capable of causing severe disease outbreaks, and; 4. Evaluate virus and vector resistance in Rubus germplasm; conduct field transmission tests to determine when viruses are being spread in the field and implement targeted control based on vector biology for management of the diseases. 5. Communicate the results: Outreach, Education and Implementation for growers, extension agents, and agricultural consultants.
1b.Approach (from AD-416):
This project aims to minimize or eliminate the impact of virus complexes in production fields through minimal pest management targeting the virus/vector combinations easiest to control, this includes the improvement of virus testing in certification programs.
This research was conducted in support of NP303 objective 3B "Characterize natural host range, reservoirs and vectors of these viruses and develop strategies to minimize their impact on production" of the parent project. Research in this project has focused on mixed virus infections in red raspberry in the Pacific Northwest and how each virus in the mixed infection contributes to severe crumbly fruit and reduced plant growth. Interactions between three viruses are being studied, Raspberry bushy dwarf virus, Raspberry leaf mottle virus and Raspberry latent virus. In cooperation with a scientist and student at USDA-ARS-HCRL, we have examined the aphid feeding response on resistant and susceptible red raspberry. Using Electro Penetration Graphing, it appears that on susceptible plants, once an aphid reaches the phloem with its stylet, it egests briefly then ingests sap from the phloem. In contrast, on aphid resistant cultivars, the aphid continues to egest after penetrating the phloem tissue and then withdraws its stylet and probes again. In this way, it appears that aphids do not ‘inject’ virus into the aphid resistant plants. In black raspberry, aphids egested and ingested on aphid susceptible plants in much the same fashion as in red raspberry. However, in aphid resistant black raspberry, the aphids seemed to not recognize when they encountered the phloem tissue with their stylets. These two separate mechanisms for aphid resistance, suggests that these could be pyramided to improve the level of resistance and decrease the chance of aphids overcoming the host resistance. Studies on the affect of aphid resistance in black raspberry on virus spread are underway.
In terms of viruses, we have now switched out focus to Rubus yellow net virus (RYNV), which was thought to be a minor virus in the Pacific Northwest when indexing was based on graft analysis. However, now that we have a polymerase chain reaction (PCR) test for RYNV we realize that graft indexing results are very different from results obtained using RT-PCR for detection. To take into account the possibility that there are isolates of RYNV that are symptomless in R. occidentalis, we tested grafted plants by RT-PCR and showed the virus was not graft transmitted in eight separate trials, in each case a positive control was graft transmitted. We repeated these tests using aphids to transmit RYNV and the non-graft transmissible isolates were also not aphid transmitted. RYNV is a member of the genus Badnavirus, and should have a circular DNA genome of about 8000 bases. We used a method called Rolling Circle Amplification (RCA), which will amplify circular DNA molecules but not linear DNA molecules. In these tests we were able to amplify RYNV from graft transmissible isolates, but not from the non-transmissible isolates. This suggests that the plants that test positive for RYNV by RT-PCR have the RYNV sequences incorporated into the host genome, rather than virus particles. The RCA amplified virus has been digested, cloned and is in the process of being sequenced.