1. Please state project objectives and what work was done this quarter to address them: CLas inhibition with antisense oligonucleotides for management of citrus greening disease Objectives: 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. 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. 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 in September 2022. The following treatments were applied as trunk injetions: FANA ASOs (LigA) and (Hel), nontarget control FANA ASO, and oxytetracycline (OTC; 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. Currently, we are processing and analyzing the data from treatments applied inSeptember 2022. A third treatment application was applied in March 2023 Fruit were harvested to assess fruit quality and yield in November 2022. After Hurricane Ian on October 11, 2022, fruit drop was quantified by installing 2×3 ft2 PVC pipe squares around each tree and counting and removing all the fruit inside the square. On November 2, 2023, fruit drop was quantified again to assess pre-harvest fruit drop. On November 10, 2022, we harvested the treatment plots based on the maturity of Hamlin fruit. Each tree was harvested entirely, and fruit was placed into individual bushel bags. Fruit were processing at the CREC Pilot Plant in Lake Alfred to obtain fruit and juice quality analyses. Fruit count, weight, and size the fruit were assessed per tree. Juice Brix/acid ratio and color were also quantified for each treatment. Results: Fruit drop. The greatest fruit drop occurred during October 2022 due to Hurricane Ian. In November, however, fruit drop was minimal. Among the trees treated with LigA-FANA, control-FANA, and Helicase-B-FANA, a total of 287, 263, and 254 fruit were collected, respectively. These treatments were the most severely affected by the hurricane and had the highest fruit drop in 2022. Trees treated with OTC had the lowest total fruit drop, followed by trees treated with insecticides only. Furthermore, the OTC-treated trees had the lowest mean fruit drop per tree, which was only 11.26 fruit/tree. In comparison, average fruit drop per tree was 17-19 fruit/tree in FANA-treated trees and 14 fruit/tree in insecticide-treated trees. The percent fruit drop was lowest in OTC-treatments (22%), followed by control-FANA (66%), LigA-FANA (69%), Helicase-B-FANA (71%), and insecticide (72%) treamtments. Fruit yield. In 2022, the highest citrus fruit yield was obtained from OTC-treated treatments (697), followed by LigA-FANA (169), control-FANA (153), insecticide (147), and Helicase-B-FANA (146) treatments. The highest mean fruit yield per tree (46 fruit/tree), occurred in the OTC treamtent, followed by Helicase-B-FANA (12.17 fruit/tree), LigA-FANA (12.07 fruit/tree), insecticide (11.31 fruit/tree), and control-FANA (10.2 fruit/tree) treatments. Citrus fruit weight (in pounds) was greatest in OTC-treated trees, with a total of 156.07 pounds, followed by LigA-FANA (34.00), insecticide (31.30), Helicase-B-FANA (28.60), and control-FANA (28.30) treated trees. The mean fruit weight per tree was the highest in the OTC treatment, with 10.40 pounds per tree, followed by LigA-FANA (2.43), insecticide (2.40), Helicase-B-FANA (2.38), and control-FANA (1.88) treatments. In addition, OTC-treated trees also produced the highest juice weight (in pounds), with a total of 78.47 pounds produced in 2022, followed by LigA-FANA (16.27), insecticide (15.19), Helicase-B-FANA (13.65), and control-FANA (13.58) treated trees. The mean juice weight per tree was the highest in OTC-treated trees with 5.23 pounds of juice per tree, followed by LigA-FANA (1.16), insecticide (1.16), Helicase-B-FANA (1.14), and control-FANA (0.90) treatments. External and internal fruit quality. The mean fruit diameter was largest from trees treated with OTC injections, (19 cm/fruit), followed by LigA-FANA (17.53 cm/fruit), Helicase-B-FANA (17.35 cm/fruit), control-FANA (16.99 cm/fruit), and insecticide-only (16.29 cm/fruit) treatments. The mean Brixº/acid ratio (15.79) was also highest in juice from OTC-treated trees, followed by Helicase-B-FANA (13.08 Brix/Acid), control -FANA (13.05 Brix/Acid), insecticide (13.02 Brix/Acid), and LigA-FANA (11.81 Brix/Acid) treated trees. No significant differences were found in the juice color among treatments. The juice from OTC-treated trees had the highest color score (30.73), followed by Helicase-B-FANA (30.35 score), control -FANA (30.34 score), LigA-FANA (30.20 score), and insecticide (30.13 score) treated trees. Overall, preliminary data from 2022 suggest that trunk injection of OTC was associated with lower fruit drop, increased yield, and improved external and internal fruit quality parameters as compared with FANA and insecticide treatments. The highest fruit production and lowest percent fruit drop were observed in response to OTC injection. Notably, fruit drop, yield, and diameter were also higher in response to FANA treatments as compared with insecticide-treated trees. No difference in Brix/Acid ratio and juice quality was observed between FANA and insecticide-treated trees. Overall, trunk injections of antibiotics appear promising for reducing fruit drop and increasing citrus yield while improving fruit quality characteristics, with FANA treatments contributing to improved fruit quality.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Acquisition and inoculation assays were replicated in fall 2022. Samples are currently being processed. 2. Please state what work is anticipated for next quarter: Objective 1:Samples from the second and third round of treatment applications will be processed and reported during the next quarter. Objective 2: Preliminary data from the second replicate of these experiments will be reported next quarter 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 will be purchased ffor the final treatment application will be purchased in the upcoming quarter. Remaining budget will be spent on field use charges and for payroll to complete sample analysis, PCR of field samples, ACP assays and colony maintenance, and fruit processing.