1. Please state project objectives and what work was done this quarter to address them:The overall objective in this proposal is to explore whether FANA oligonucleotides, which are synthetic RNA-based synthetic molecules that mimic DNA and inhibit bacterial gene expression can be used to mitigate CLas in infected trees.The first objective of this project is to s 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. We have initiated field trials with laboratory-vetted FANAs in a research grove at the UF Citrus Research and Education Center in March 2022. Treatments are applied to 10-year-old, CLas-infected Valencia trees of a standard size and CLas titer. FANA ASOs complementary to two CLas essential genes and a scramble sequence control are synthesized by AUM LifeTech, initiated in December 2021. Antibiotic application (Fireline) and insecticide-only treatments are applied to trees as control treatments. Each treatment is applied to 15 trees in 1-acre plots, replicated five times in a randomized complete block design. Treatments are applied using microinjection of dosages determined in our previous greenhouse assays.Prior to treatment 1 (T1), 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. PSamples are removed from the same branches as the T0 samples after 2, 7, 30, 60 and 90 days. The second objective is to evaluate FANA antisense oligonucleotide targeting CLas in order to reduce vector transmission. Field assays with psyllids are conducted to evaluate the efficacy of FANAs for inhibiting Las transmission by ACP. Single females from uninfected laboratory cultures are caged on young leaf growth (flush) of treated or control infected trees for oviposition. Each treatment is replicated ten times on individual trees. This experiment was initiated in March 2021, as outlined in our proposal timeline. The effect of FANA treatments on acquisition of CLas will be assessed by comparing the CLas titer in ACP caged on citrus trees before and after treatments and across time. 2. Please state what work is anticipated for next quarter: Objective 1: PCR of these leaves and leaves collected post treatment will be conducted during Q2 of this project to monitor the effect of the FANA ASOs on the CLas titer of each tree. Objective 2: Initial results after 1 treatment 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. Personnel spending has been low to date, as December 21- January 22 were primarily spent designing and synthesizng FANA molecules for the experiment.