1. Please state project objectives and what work was done this quarter to address them: Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy in a field trial. Field trials were conducted in research groves at the UF Citrus Research and Education Center. Treatments were applied to 10-year-old, CLas-infected ‘Hamlin trees of a standard size and CLas titer. The following treatments were as trunk injetions in May 2022: FANA ASOs complementary to CLas NAD-dependent DNA ligase gene (LigA) and CLas DNA B- helicase gene, nontarget control FANA ASO, oxytetracycline (Fireline). Injected treatments were compared with an insecticide-only control treatments. Each treatment was applied applied to 15 trees in 1-acre plots, replicated three times in a randomized complete block design. Treatments were applied to both sides of the tree canopy using microinjection of dosages determined in our previous greenhouse assays. All FANAs dosages were 625 ppm per tree. To monitor the effect of treatments on trees CLas titers as assessed 0, 2, 7, 30, 60 and 90 days post-treatment by qRT-PCR analysis. A second replicate of the treatements in this experiment was applied in September 2022. Fruit were harvested to assess fruit quality and yield in November 2022. Results: CLas titers were initially similar across treatments, with all trees infected. CLas titers declined in FANA-treated and oxytetracycline -treated trees 30d after treatments were applied. CLas titers were lowest in trees treated with LigA-FANA or oxytetracycline at 30d and 60d post treatment. Trees treated with helicase-B-FANA also exhibited a reduction in CLas titer comparable to oxytetracycline treaments on 30d and 90d post-treatment. Overall, trees injected with ligA-FANA, helicase-B-FANA, or oxytetracycline had significantly lower CLas titers at 30, 60, and 90 days post treatment compared to the control treatments (insecticide or non-target FANA). Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Acquisition assay.Acquisition assays were replicated this quarter. Samples are currently being processed. Inoculation assay. A subsample of 10 ACP per treatment were collected from treated trees was transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP were enclosed on plants for inoculation feeding for seven days. Inoculated plants were sampled at 30, 60, and 90 days after inoculationsto assess the development of CLas infection following ACP exposure. For this assay, each treatment was replicated five times on individual trees. Results: Forty percent of trees exposed to ACP from LigA-FANA and Helicase-B FANA treatments were infected with CLas 30 days after inoculation, compared with 20% trees exposed to ACP from insecticide treatments. Sixty days after inoculation, 40-80% of trees were infected in the LigA-FANA, B-Helicase-FANA, scramble-FANA, and insecticide treatments Ninety days post-inoculation, a similar pattern was observed; however, plants exposed to ACP from insecticide-treated trees were 100% positive for CLas infection. Plants inoculated by ACP from oxytetracycline-treated trees were the only treatment to remain CLas-free after 30, 60, and 90 days post-inoculation.These initial results suggest that ACP collected from FANA-treated trees are less likely to inoculate healthy citrus. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from the second round of treatment applications through day 60 will be processed during next quarter. Tree health and fruit quality/yield data will be reported next quarter. Objective 2: Acquisition assays were replicated and results will be reported next quarter. 3. Please state budget status (underspend or overspend, and why): Our budget is on track for the project. FANA treatments, which are the largest portion of the budget, have been purchased for the experiments. Remaining budget will be spent on field use charges and for payroll to complete sample analysis, ACP assays and colony maintenance. Microinjectors needing replacement will be purchased next quarter for injections in April.