In this study, we will pursue the following specific objectives:1) GFP labeling of Candidatus Liberibacter asiaticus. 2) Elucidation of plant-Las interaction through real-time monitoring of Las movement and multiplication in planta using GFP labeled Las. 3) Investigate the effect of different control approaches on the dynamic population of Las in planta using GFP labeled Las. Previously, the reporter plasmid, pBAM1::R-PgyrA-GFP, composed of Tn5 and narrow host-range origin was constructed and therefore the GFP gene can be inserted into the genome of bacteria. However, it was only successfully transferred into a genome of Pseudomonas fluorescence with low transformation efficiency and failed with other bacteria including Escherichia coli DH5a, Sinorhizobium meliloti Rm1021, and Liberibacter crescens BT-1. Recently, pDH3::PgyrA-GFP was constructed which has a wide bacterial host range replicon, repW, but cannot be inserted into a genome. Transformation of E. coli by PEG mediated method with pDH3::PgyrA-GFP showed high transformation efficiency (~2 x 104 CFU/µg of DNA) than with previous reporter plasmid (failed). Following application with L. crescens BT-1 by electroporation was also successful (1.9 x 103 CFU/µg of DNA). Transformants and the GFP expression in L. crescens BT-1 were confirmed by PCR and fluorescent microscopic analysis, respectively. As L. crescens is a phylogenetically closest species to Ca. L. asiaticus, there is a possibility that pDH3::PgyrA-GFP would be useful for GFP labeling of Ca. L. asiaticus. We have further confirmed the Lcr-GFP using western blot. The GFP plasmid is being used to transform Las. To facilitate Las transformation, we have tested multiple novel methods of culturing. Las population was observed to decrease at the beginning, and increase slowly. Repeated experiments show similar pattern which suggest we might be able to acquire enough Las cells for transformation after further optimization. We are testing new methods for culturing Las. Especially, we are testing co-culturing Las with citrus tissue culture and psyllid tissue culture. Currently, we are in the process of establishing a pure psyllid cell culture. We have used two approaches to label L. crescens. Preliminary data showed one approach works for Las in vitro. We are testing whether we can label Las in vivo and observe its movement. 2) We have conducted Las movement and multiplication in planta based on qPCR method. We have tested approaches to prevent Las movement in planta. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. 3) We have been testing the effect of different control approaches including application with bactericides. One manuscript entitled: “Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics” has been published by Phytopathology. In addition, based on the movement of Las in planta, we have developed a method for targeted early detection of Las before symptom expression. A manuscript entitled Targeted Early Detection of Citrus Huanglongbing Causal Agent ‘Candidatus Liberibacter asiaticus’ Before Symptom Expression has been published by Phytopathology. We determined the in planta minimum inhibitory concentration of oxytetracycline against Candidatus Liberibacter asiaticus effective for control of citrus Huanglongbing which has been accepted with revision by Phytopathology. In addition, we further characterized the movement of Las in planta and the treatment effect of antibiotics using trunk injection and foliar spray. Two manuscripts are being prepared.