Project Number: 2072-22000-046-021-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2022
End Date: Jul 31, 2023
1. Optimize the infectious clone to efficiently infect blueberry and screen major cultivars for susceptibility to the virus. 2. Survey blueberry production areas. 3. Test aphid species for their ability to vector the new virus.
Objective 1: Cultivar susceptibility will be assessed using the already developed infectious clone. The benefit of such an approach is that we will obtain single infections and be able to study the range of symptoms solely attributed to the virus, something that cannot be guaranteed if using grafting or vectors. Tissue culture plants, tested free of all pathogenic blueberry viruses, will be purchased and used in the optimization of agroinfiltration, slash inoculation or bombardment of the infectious clone. We aim to use different vector/agrobacterium strains (pCB302-3, pJL 89, pORE-E4)/ pCB302-3, pJL 89, pORE-E4) in all possible (9) combinations to optimize the process. If agroinfiltration and slash inoculation prove unsuccessful with all aforementioned combinations we will move to bombardment which, even though not practical, will allow for successful delivery of the vector to blueberry plants. Plants will be kept in growth chambers until establishment of systemic infection. Once the virus is systemic, as validated by virus detection by RT-PCR in systemic leaves, newly emerged shoots will be sent to Corvallis, OR for graft inoculation onto major cultivars including but not limited to Aurora, Berkeley, Bluecrop, Bluegold, Chandler, Draper, Duke, Elliott, Legacy, Liberty, Last Call, Titanium, Top Shelf. We feel that symptom expression should be done in Oregon as it has optimal environmental conditions for symptom development. If there are cultivars that are not successfully inoculated by any of the methods described, a possible indication of immunity to the virus, we will graft them at least three times to confirm immunity. Objective 2: One thousand blueberry plants have been collected from blueberry growing regions of the U.S. An RT-PCR assay developed by the University of Arkansas will be utilized to screen all samples. Total RNA has been extracted and RT-PCR will be performed in-house. Positive reactions will be validated by cloning and sequencing amplicons. Objective 3: The most abundant aphid species in the Pacific Northwest, presumably the green peach aphid, Myzus persicae, and blueberry aphid, Fimbriaphis fimbriata, will be collected from areas where the virus is known to occur. After molecular confirmation of the phenotypic identification, nymphs will be collected immediately after emergence – carlaviruses are not transmitted transovarially- and used for the establishment of virus-free colonies. Plants from Objective 1 with the highest virus titer (as assessed by semi-quantitative RT-PCR) will be used as source material in transmission studies to be conducted in year 2 of this study. Additionally, common aphids on blueberry in other production regions will be tested as vectors in year 2, as an example, Illinoia pepperi, in Michigan.