2012 Annual Report 1a.Objectives (from AD-416): Objective 1: Develop and use assays to characterize the interactions of exotic citrus pathogens with their hosts or vectors. Objective 2: Determine the etiology of exotic diseases of citrus and the diversity of exotic citrus pathogens. 1b.Approach (from AD-416): This research project is focused on huanglongbing (HLB) and the extremely invasive pathogen Ca. Liberibacter asiaticus and another potentially invasive but uncharacterized pathogen, a presumed virus that causes citrus chlorotic dwarf (CCD) in Turkey. The pathogen has not been described, but it is transmitted by a whitefly already widespread in the United States and thus has very high potential for invasiveness. We will conduct basic and applied research which will lead to improved understanding of the biology and etiology of these diseases and provide the basis for improved diagnostic and research methods. Microarray studies of sweet orange trees infected with Ca. Liberibacter asiaticus and other phloem-limited pathogens will lead to the identification of novel host genes associated with general disease responses triggered by phloem-limited pathogens and specific disease responses triggered by Ca. Liberibacter asiaticus. Recombinant antibodies developed by bacteriophage display will be used to develop diagnostic procedures for Ca. Liberibacter asiaticus and as research tools. We will analyze data obtained from deep sequencing technology to characterize the virus causing citrus chlorotic dwarf. This information will be used to develop PCR and serologically based assays for this pathogen. The products of this research project will improve both scientific knowledge of invasive pathogens of citrus and provide novel assays useful or their detection and management. 3.Progress Report: We have begun the analysis of the microarray expression data set using statistical software packages ‘R’ and ‘BioConductor’. We have also isolated RNA from citrus trees infected with Citrus chlorotic dwarf virus. Small RNA produced by the host in response to the virus has been sequenced using Illumina deep sequencing technology. Bioinformatic analysis of the sequence data has provided identification of novel virus associated with Citrus chlorotic dwarf disease. Final sequence assembly and annotation are underway. Further research on Xylella fastidiosa in sweet orange seed is ongoing, funded by a grant from USDA APHIS CPHST. We also have also continued work on a project funded by the Florida citrus industry to develop antibodies against the citrus greening pathogen. We have made a ‘library’ of several million antibodies against infected insect extracts and continue the process of screening the library to identify individual antibodies with desired specificity. Please see the report for Project 1275-22000-251-00D for further information.