The purpose is to evaluate the control effect of bactericides via trunk injection.Objective 1. 1.1. Determination of the in planta minimum bactericidal concentrations (MBCs) of bactericides against Las We developed a new method for evaluating the effects of oxytetracycline (OTC) treatment on Las titers in planta, and determined the relationship between OTC residue levels and control levels achieved for Las using mathematical modeling in greenhouse and field experiments. In greenhouse, OTC injection at 0.05 g/tree decreased Las titers to an undetectable level (Ct value = 36.0) from 7 to 30 DPA, and produced a residue level of OTC at 0.68-0.73 µg/g fresh tissue over this period. In the field, OTC injection at 0.50 g/tree resulted in the decline of Las titers by 1.52 log reduction from 14 to 60 DPA, with residue levels of OTC at 0.27-0.33 µg/g fresh tissue. In both trials, a first-order compart model of OTC residue dynamics in leaves of trunk-injected trees was specified for estimating the retention of effective concentrations. Furthermore, nonlinear modeling revealed significant positive correlations between OTC residue levels in leaves and the control levels for Las achieved. The results suggested that the minimum concentration of OTC required to suppress Las populations in planta to below the detection limit is 0.68 and 0.86 µg/g, and the minimum concentration of OTC required for initial inhibition of Las growth in planta is approximately 0.17 and 0.215 µg/g in leaf tissues under greenhouse and field conditions, respectively. This finding highlights that a minimum concentration of OTC should be guaranteed to be delivered to target Las in planta for effective control of citrus HLB. This study has been published by Phytopathology in a manuscript entitled: The in planta effective concentration of oxytetracycline against Candidatus Liberibacter asiaticus for suppression of citrus Huanglongbing. In addition, we evaluated the inhibitory activity of streptomycin (STR) against Las in a greenhouse experiment. Citrus trees were trunk-injected with STR, and leaves were inspected for Las populations and STR residues using qPCR and HPLC assays respectively, at various times after STR injection. Assays for Las titers and STR concentration in leaf samples from field trials are also ongoing. We are summarizing the data for publication and presenting the information to citrus growers. 1.2. Effect of bactericides via trunk injection on citrus HLB disease progression, tree health, yield and fruit quality in different aged trees with a different disease severityThe field experiments were performed at four different groves on different aged trees (2, 3, 7 and 20 years old) with a different disease severity. For all the four field trials, the injection treatment applications were completed by the end of April 2019. The first application of spray treatments were completed during spring flushing in February or March 2019, and the second applications were conducted in late June to early July 2019. Leaf samples have been collected from the treated trees at the following time points: 0 (pre- injection), 7, 14, 28 days, 2, 4, and 6 months after treatment. The estimation of Las titers in these leaf samples are ongoing with qPCR assays. The first estimation of HLB disease severity and growth performance (height, trunk diameter, and canopy volume) of immature trees after treatment were performed on May, 2019 (three months after the injection) and continued in a 3-months interval. Fruit yield and quality data were collected in January 2020.Objective 2. 2.1. Examination of dynamics and residues of bactericide injected into citrus and systemic movement within the vascular systemLeaf and root samples have been collected from OTC or STR treated trees in the Avon Park grove at the following time points:0 (pre- injection), 7, 14, 28 days, 2, 4, 6 and 8 or 9 months after injection. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples were determined by HPLC assays. 2.2. Determination of the residue contents of bactericides in fruit and juice in each harvestFruit samples were collected during harvest in January 2020. The samples are being processed for OTC or STR extraction, and the concentrations of OTC and STR in these samples are being determined by HPLC assays.2.3. Analysis of degradation metabolites of bactericides injected into citrus treesLeaf samples were collected from OTC or STR injected trees in the Avon Park grove at two and four months after treatment for the analysis of the degradation metabolites of the bactericides. The samples are being processed for the extraction of the degradation metabolites.Objective 3. 3.1. Greenhouse assays of the effect of bactericides via trunk injection on Las acquisition by ACPThis assay will be initiated in the spring of 2020.3.2. Field assays of the effect of bactericides via trunk injection on Las acquisition by ACPThis assay will be initiated in the spring of 2020.Objective 4. 4.1. Monitoring resistance development in Las against bactericidesLeaf samples for this test have been collected from 5 trees injected with OTC and 5 trees injected with STR at the highest doses in each of the three groves at six and nine months after the injection. PCR-sequencing analysis on Las 16SrRNA gene showed there was no mutation compared with the reported sequence at the 6-month samples. 4.2. Evaluation of potential side effects of trunk injection of bactericides We evaluated possible phytotoxity caused by OTC or STR in immature trees (3-year old Valencia) from one week to one month after injection. The trees were be examined for the following symptoms: fruitlet drop, fruit drop, quantity of leaf drop, non-insect related leaf rolling, and leaf discoloration. There was no significant difference in fruitlet drop, fruit drop, quantity of leaf drop, or non-insect related leaf rolling between OTC or STR treatment and untreated control. About 20% (3 out of 15) trees injected with OTC or STR at the highest dose (2.0 g/tree) showed leaf discoloration (yellowing) on some young shoots. These phytotoxicity-like symptoms disappeared at 6 months post injection. There was no infection symptom by Phytophthora in the area surrounding drilling sites (injection holes), probably due to the application of the fungicide Ridomil gold immediately after drilling.In 7-year old Hamlin trees in the Auburndale grove, three trees injected with STR at the highest dose (3.0 g/tree) showed leaf discoloration (yellowing) on some young shoots, a possible phytotoxitic effect. These phytotoxicity-like symptoms disappeared at 6 months post injection. Other treated trees all showed normal growth.We are continuing the surveys for potential side effects.