. 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. Our working hypothesis is that CLas-specific FANAs can be delivered using microinjection developed for RNAi-based technologies to reduce CLas in infected citrus trees. Field trials with laboratory vetted FANAs 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. AUM LifeTech designed and synthesized FANA ASOs to be complementary to two CLas essential genes. As a negative control, a FANA ASO was designed as a scramble sequence with no complementarity with any citrus gene. Antibiotic application (Fireline – Oxytetracycline) and insecticide-only treatments were applied to trees as control treatments. Each treatment was 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. The first replicate of this experiment was conducted from May through August and consist of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas L-FANA, and CLas B H-FANA. All FANAs dosages were 625 ppm per tree. Prior to treatment, four leaves were removed from each tree, two from each side of the apex of the tree and two from each side of the base of the canopy, for initial titer (T0) using quantitative real-time polymerase chain reaction (qPCR) assays. To monitor the effect of the FANA ASOs on the CLas titer of each tree, four leaves samples were removed from the same branches as the T0 samples after 2, 7, 30, 60 and 90 days. The post-treatment CLas titer (TF) were calculated by qRT-PCR analysis, at each time. Leaf samples were run in duplicates, and the relative quantities of CLas in threes were calculated based on the comparative cycle threshold 2-..Ct method. Results: In this report, information regarding the first application of treatments is presented. At T0, CLas titters in trees were similar across treatments. Two and seven days after treatment were applied, CLas titers in trees fluctuated; and higher CLas titers were observed in FANA treated trees compared to antibiotic and insecticide treated trees. No differences were found in CLas titers among treatments 30 days after treatments were applied. However, CLas titers were lower in trees treated with L-FANA and titers were comparable with those trees treated with antibiotics 60 days after treatments were applied. Overall, trunk-injections of L-FANA and antibiotic treatments significantly reduced CLas titers in trees as compared with insecticide only or control FANA treatments. A second replicate of this experiment will be performed in September of 2022 to confirm our observations. Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Our working hypothesis is that CLas will be specifically inhibited in psyllids by using CLas-specific FANAs, resulting in a reduction in Las acquisition and transmission by ACP in a field setting. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in June 2022. Psyllid nymphs, who develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees were compared with acquisition from untreated infected trees, using the treatments described in Obj.1. Seven days after treatments were applied, ten ACP (five males and five females) from uninfected laboratory cultures were caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment was replicated three times on individual trees. Following oviposition (seven days after), ACP adults (P1) were collected and preserved for CLas detection. Egg clutches were left on trees enclosed in mesh sleeves. After nymphs reached the adult stage (15 days after), psyllids (F1) and leaves from test plants were collected. The effect of FANA treatments on acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees. Results: The results from a bioassay assessing acquisition of CLas following FANA treatments showed that ACP adults (P1) and offspring (F1) were able to acquire CLas across treatments 7 and 15 days following treatments, respectively. The lowest CLas titers were observed in P1 and F1 ACP feeding on antibiotic-treated trees; followed by L -FANA and H-FANA treatments. The highest CLas titers were observed on ACP adults and offspring feeding on Scramble-FANA and insecticides only treated trees. Overall, L -FANA, H-FANA, and antibiotic treatments reduced CLas acquisition by ACP as compared with scramble-FANA and insecticide treatments. A second replicate of this experiment will be performed in September 2022. 2. Please state what work is anticipated for next quarter: Objective 1:The second treatment application is planned for this period. Samples from the second round of treatment applications through day 30 will be processed during quarter #3. Tree health and fruit quality/yield will be assessed in quarter # 4. Objective 2: Acquisition assays will be replicated and partial results with P1 and F1 adults will be reported in quarter #4. Inoculation assays will also be completed in quarter #4, with samples collected for CLas detection. If rootstocks are used for inoculation, plants will be held for development of CLas infection and symptom development. 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 for sample analysis, ACP assays and colony maintenance, research plots, and personnel. Microinjectors needing replacement will be purchased next quarter.