1. Please state project objectives and what work was done this quarter to address them:Objectives of this project are to determine the phytotoxicity of OTC and other potential partner antibiotics 1) by a citrus leaf assay in the lab, 2) by a greenhouse assay in seedling trees, and 3) to compare OTC with other antibiotics’ efficacy against CLas on mature trees in the grove. This quarter (Q1), we purchased most of the proposed antibiotics, except zhongshengmycin, which was not available from the manufacturers in China. Therefore, we replaced this antibiotic with another antibiotic, nalidixic acid, which was shown to be effective on various gram-negative bacteria. We have successfully completed tasks pertaining to Objective 1, where we screened all experimental antibiotics and imidacloprid, along with OTC to evaluate their phytotoxicity to citrus. In these lab experiments, we used a detached-leaf assay to assess ‘exaggerated’ concentrations quickly and to document any potential phytotoxicity. In repeated (at least three times) experiments, only oxolinic acid and nalidixic acid at 600 ppm caused light phytotoxicity, including petiole and midvein necrosis; whereas other antibiotics, namely OTC, kasugamycin, streptomycin, gentamicin and validamycin A, all at 600 ppm, and an insecticide imidacloprid at 800 ppm, did not show any phytotoxicity. Further evaluation of these two phytotoxic antibiotics revealed that the solvent used for dissolving them per manufacturer’s recommendations (NaOH 0.01M) was a potential culprit behind this phytotoxicity. Therefore, we concluded that none of the tested antibiotics and imidacloprid were phytotoxic to citrus leaves at these exaggerated concentrations.Next, we conducted similar phytotoxicity assays, this time on greenhouse-grown healthy Valencia seedlings in the greenhouse (Objective 2). Here, all the treatments we used in the leaf assays from above were used in 10X more exaggerated concentrations (i.e., 6000 ppm) per tree and additional 3000 ppm for gentamicin after seeing high phytotoxicity in that treatment in the first repetition. We used the drill-based trunk injection (Chemjet) technique to deliver these treatments from the seedling trunk. All treatments were taken up by the trees without any issues except for the imidacloprid (8000 ppm), which was in a formulation that was not recommended for trunk injection. In the subsequent trial, we replaced this formulation with the brand (Xytect) that was formulated specifically for the trunk injection of forest and ornamental trees. No uptake issue for this new formulation was observed. Regarding phytotoxicity in the greenhouse, at least one out of five trees per treatment in repeated trials showed some level of phytotoxicity–particularly in the drill site on the trunk–including on the trees that were injected with water. This suggested that drilling a hole in the trunk of young (1.5-2 years old) seedlings is very damaging; 10-70% of the drill sites showed signs of oozing, regardless of the treatment. Nevertheless, only a small number of trees showed some phytotoxicity on the leaves, including dieback, leaf discoloration, defoliation, and leaf curling. Among them, gentamicin was the most phytotoxic to citrus at 6000 ppm, as three out of five treated plants showed severe dieback, and eventually, those plants died. However, gentamicin at 3000 ppm was not as phytotoxic, and showed only leaf curling on 60% of the treated plants, with none of the treated plants dying. Much milder symptoms, such as leaf mottling, chlorosis, and vein yellowing, were observed in only a few plants treated with kasugamycin and streptomycin at 6000 ppm. None of the other antibiotics showed severe phytotoxicity, but only occasional leaf chlorosis or leaf curling, which did not affect the trees’ overall health. Additionally, Xytect–injectable imidacloprid–caused severe phytotoxicity, including oozing on the treatment sites (40%), dieback of the plants (40%), leaf discoloration (40%), defoliation (60%) and leaf curling (80%). 2. Please state what work is anticipated for next quarter:1) Greenhouse assays will be repeated one or two more times to have better insight into the phytotoxicity of these treatments. 2) We will finalize the phytotoxicity experiments, analyze data, and make decisions on what treatment and in what concentration they can be injected into citrus trees before moving onto field experiments. 3) The grove site with mature Valencia and Hamlin trees was identified at the SWFREC experimental grove. We have started taking measurements and disease ratings of trees (Time zero; T0), as well as labeled them to prepare for the upcoming trunk injection trials (Objective 3). Trees are flushing now, and we believe that at the mid-March or end of March, we will be injecting those trees. 3. Please state budget status (underspend or overspend, and why): The budget is spent as expected.