The purpose of this project is to reveal the mechanisms of bactericide uptake and transport in citrus plant and establish a theoretical basis for developing technologies to improve the efficacy of bactericides, which is helpful to provide potential solution to the development of effective chemotherapeutic tools for HLB management. Achieving this outcome will require progress in the following three tasks: (1) to compare the delivery efficacy of bactericides with three application methods (foliar spraying, truck injection, and root administration) based on the uptake and dynamic movement/distribution of the bactericide within the tree; (2) to clarify the systemic movement and transportation mechanisms of bactericides within the phloem of tree; and (3) to investigate the effects of citrus variety and age on the delivery efficacy of bactericides. This project requires a combination of greenhouse studies and field trials. Prior to conducting these experiments, a sensitive and accurate quantifying method of bactericides (oxytetracycline and streptomycin) in citrus tissues is needed. This project officially started on December 1, 2019. This is the 2nd quarterly progress report covering 3/01 to 05/31, 2019. During this period we have started and/or completed the following work/research tasks: 1) We conducted a literature search for methods that can be used for analyses of oxytetracycline and streptomycin in plant samples and the methods we found in literature are adequate for analysis of oxytetracycline or streptomycin only in processed food such as fruit gel. They are not reliable for determination of oxytetracycline or streptomycin in plant samples, because of matrix effects that result in poor extraction efficiency. 2) In this project, we need a reliable method for simultaneous determination of both oxytetracycline and streptomycin in citrus plant tissue samples, but it is not available in literature. Therefore, we designed a method based on information in literature and our previous experience in the extraction of other antibiotics in plant samples for simultaneous analysis of oxytetracycline and streptomycin in citrus plants, especially we replaced methanol with acetonitrile and used lower concentration of formic acid for the extraction of antibiotics in citrus plant tissue samples. 3) Laboratory tests demonstrated the potential and feasibility of the newly designed method for simultaneous analysis of oxytetracycline and streptomycin in citrus plants, but matrix interference with antibiotic analysis was still significant. 4) To further improve this method, a solid-phase extraction (SPE) method was developed to clean up sample matrix, and analytical conditions of instrumentation were modified by changing the gradient profile of LC and lowering the operation temperature of MS/MS. These modifications substantially improved instrumental sensitivity for detecting oxytetracycline and streptomycin plant samples. 5) This newly developed method has now been successfully applied to the simultaneous detection of oxytetracycline and streptomycin in citrus leaf samples. The work planned for the next quarter:The major goals of research for the next six months are to evaluate the delivery efficacy of bactericides within citrus trees based on three different application methods (foliage spraying, trunk injection, and root administration). The following research will be conducted in the 3rd quarter: 1) acquisition and culture of citrus seedlings in greenhouse; 2) foliar spraying of streptomycin or oxytetracycline and periodic sampling; 3) sample extraction and instrumental analyses; and 4) preparation of materials for experiments with application methods of trunk injection and/or root administration.