1. Please state project objectives and what work was done this quarter to address them: The overall goal of the proposed research is to test the effectiveness of Frateuria defendens (Frd) in reducing HLB damage in FL citrus. The original proposal was revised for a 1-year feasibility study. Prior to the start of the funding period, we prepared our facility and cleared all regulatory hurdles to allow import of and work with the bacterium in FL under a valid APHIS permit. Due to the regulated nature of the organism, all experiments are being conducted in a greenhouse.Objective 1. Determine the optimal application method of Frd to citrus trees.To optimize the application process, the Israel team conducted a series of in-lab and in-field experiments to optimize the culturing process and determine optimal spray concentrations and time of survival of Frd. During several virtual meetings, methods and outcomes were discussed among the Israel and FL team, and protocols were established. Upon start of the funding period, culturing methods were successfully implemented in the Albrecht lab. We also established real-time polymerase chain reaction methods to detect Frd in planta. At the same time, methods were established and successfully implemented to detect viable bacteria in plants after application. Objective 2. Determine if Frd pre-treatment can prevent trees from getting infected with Candidatus Liberibacter asiaticus (CLas).Due to the short duration of this grant and the complications associated with creating a homogenous population of infected plants, we are first testing efficacy of Frd in infected symptomatic plants (curative). Should Frd show efficacy in restoring health of infected plants, we will initiate additional longer-term pre-treatment (preventative) experiments.Prior to the start of the grant, we prepared plant material to be used for greenhouse testing. For this, we grafted buds from HLB-symptomatic field plants (Valencia sweet orange) onto UFR-5 rootstock liners to create a population of CLas -infected grafted Valencia trees for experimentation. Six months after grafting, all plants were tested by PCR for CLas presence and titer levels. Plants were divided into different populations based on titer levels, disease severity, and size to be used for either spray experiments (smaller plants) and larger plants (injection experiments). Plants that were PCR negative for CLas were not included.In December, the first experiment was initiated to test efficacy of spray applications of Frd in restoring plant health and reducing CLas titers. Frd is being was sprayed in biweekly and monthly intervals at the concentrations determined optimal during the pre-grant stage. Control plants are receiving culturing medium only. Leaves are collected regularly to assess CLas and Frd titers and horticultural variables (plant height, stem diameters, leaf number, etc.) are being measured to assess efficacy of treatments on plant health. 2. Please state what work is anticipated for next quarter: A second experiment using the larger set of infected plants is in preparation to examine efficacy of Frd by trunk injection; we will continue optimizing the injection method before commencing with the experiment. We will continue with the spray experiment, collecting horticultural information and determining CLas and Frd titers. 3. Please state budget status (underspend or overspend, and why): Approximately 25% of funds have been spent.