1. Please state project objectives and what work was done this quarter to address them: Objectives:.1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. 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 to reduce vector transmission. Our working hypothesis is that CLas will be inhibited explicitly in psyllids by using CLas-specific FANAs, resulting in reduced CLas acquisition and transmission by ACP under field conditions.Methods:Objective 1. Screen FANA antisense oligonucleotide targeting CLas for efficacy under field conditions. 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 complementary to two essential CLas genes: the CLas NAD-dependent DNA Ligase gene (LigA) and the CLas DNA B-Helicase gene. 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 positive and negative control treatments, respectively. 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 spring and fall 2022 and spring 2023. It consisted of five treatments: untreated control (insecticide-only), oxytetracycline control (1.56 g of Fireline per tree), Scramble Control-FANA, CLas LigA-FANA, and CLas B Helicase-FANA. All FANAs dosages were 625 ppm per tree. Update: This report provides an update on the effect of FANA ASOs on CLas infection in trees, tree growth, and yield after two years of the study. In the fall of 2023, a fourth injection of treatments was performed. CLas infection remained significantly low in trees treated with oxytetracycline trees from day zero and up to 90 days following application. LigA-FANA-treated trees showed a significant reduction in CLas infection at day 7- and 30 days following application, and results were comparable with oxytetracycline-treated trees. However, at 45, 60, and 90 days following application, FANA-treated trees showed similar CLas infection levels to those in the insecticide-treated (negative contrl) trees. At the end of the experiment, ten dead trees were recorded: 2-3 trees in FANA-treated plots (13-20%, n = 15), three trees in insecticides-treated trees (20%, n = 15), and none in oxytetracycline-treated trees. Additionally, three health parameters (Canopy and CGU) were recorded at the end of the experiment to assess the cumulative effect of treatments on trees. These results are currently being analyzed and will be provided in the final report. 2023 Harvest season data:Fruit yield. In 2023, trees treated with oxytetracycline yielded 5 – 20 times more fruit than trees in FANA ASO and insecticide-treated plots, respectively. Among these treatments, trees treated with oxytetracycline yielded significantly more fruit per tree (40.4 fruit/tree) than the other treatments evaluated. The highest total fruit weight was produced by trees treated with oxytetracycline with a total of 124.27 lbs., followed by Helicase-B-FANA (9.24 lbs.), Control-FANA (8.1 lbs.), LigA-FANA (3.02 lbs.), and insecticides-only (1.47 lbs.) treated trees. The mean fruit weight per tree was significantly higher in oxytetracycline-treated trees, with 8.29 lbs. of fruit weight per tree compared with 0.3-1.32 lbs. of fruit weight produced per tree observed from the rest of the treatments.Fruit drop. Fruit drop was successfully recorded prior to the harvest of the plots. Results are currently being processed and analyzed and will be included in the final report. Juice yield and quality analyses. Oxytetracycline-treated trees produced the highest juice weight, with a total of 62.65 lbs. produced in 2023, followed by Helicase-B-FANA (3.97 lbs.), Control-FANA (3.7 lbs.), LigA-FANA (1.17 lbs.), and insecticides-only (0.65 lbs.) treated trees. The mean juice weight per tree was significantly higher in oxytetracycline-treated trees, with 4.18 lbs. of juice per tree compared to 0.13-0.57 lbs. of juice produced by the rest of the treatments. Moreover, oxytetracycline-treated trees also had a significantly higher Brixº/acid ratio, averaging 12.5 Brix/Acid in the juice compared to 9.72-10.62 Brix/Acid achieved by the rest of the treatments. Juice color parameters were improved in 2023 compared to 2022. The juice from oxytetracycline-treated trees had the highest color score of 31.87, followed by LigA-FANA (31.2 score), insecticides-only (31.2 score), Helicase-B-FANA (31.03 score), and Scramble-FANA (30.98 score) treated trees. However, no statistically significant differences among treatments were found in the juice color.Objective 2. Evaluate FANA antisense oligonucleotide targeting CLas to reduce vector transmission. Acquisition assay. Field assays with psyllids were conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP in the spring and fall of 2022 and spring and fall of 2023. Psyllid nymphs, which develop on immature leaf tissue, acquire CLas more efficiently than adults; therefore, acquisition of CLas from FANA-treated infected citrus trees was 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 the acquisition of CLas was assessed by comparing the CLas titer in P1 and F1 ACPs caged on treated and untreated citrus trees.Overall, the fewest infected ACP adults were collected on oxytetracycline-treated trees, followed by LigA-FANA-treated trees. For emerging ACP nymphs, CLas infection was only statistically reduced on trees treated with oxytetracycline compared to the control. The last replication i scurrently finishing. Results are being processed and analyzed and will be added to the final report. To evaluate incoculation, a subsample of 10 ACP per treatment was collected from treated trees and transferred to uninfected citrus seedlings in an insect-proof greenhouse. ACP adults were enclosed on plants for inoculation feeding for seven days. The last replication of the experiment was performed in the first quarter of 2024. Results are currently being analyzed and will be added to the final report. 2. Please state what work is anticipated for next quarter:The remaining fruit and juice quality data will be compied analyzed. All acquisition and inoculation assays will be completed, final samples processd, and data will be analyzed in the next quarter. 3. Please state budget status (underspend or overspend, and why): The budget spending is on track as anticipated. 4. Please show all potential commercialization products resulting from this research, and the status of each: Not applicable at this time. THis project is evaluating registered and available products.