2012 Annual Report 1a.Objectives (from AD-416): 1. To develop ultra sensitive Taq-Man PCR diagnostic system for Improve detection of Ca. Liberibacter asiaticus. 2. To design a Ca. Liberibacter genus-specific real-time PCR technology for general detection of Candidatus Liberibacter species. 1b.Approach (from AD-416): 1. Cloning, sequencing and characterizing genomic sequences of citrus Candidatus Liberibacter species using novel molecular technique. 2. Design and validate sensitivity and specificity of diagnostic systems. 3.Progress Report: This trust agreement supports objective 1.B of the parent project. The goal of the project is to develop improved diagnostic systems for reliable and sensitive detection of bacterial species associated with citrus huanglongbing and potato zebra chip disease. Two new diagnostic systems have been designed and validated. The first is called “Single Tube Dual Primer Taq-Man Polymerase Chain Reaction (STDP-PCR)” which provides ultra sensitivity for detection of citrus HLB. The second detection system is named, “Fluorescent dye-based High Resolution Melting Curve Real-Time Polymerase Chain Reaction (FHRMA-PCR). This system can be used for detection and differentiation of up to four Liberibacter species, including the three known species associated with citrus HLB (‘CL asiaticus’, ‘CL africanus’ and ‘CL americanus’) and one species associated with potato zebra chip disease (CL solanacearum’). Both systems have been validated using DNA samples collected from plants and insects. Both detection systems provide high throughput capabilities suitable for large scale, year around quarantine screening and epidemiological studies.