2012 Annual Report 1a.Objectives (from AD-416): 1) To conduct molecular characterization and determine the classification groups for Phytoplasma from California and in the exotic disease collection in Beltsville, MD. 2) To develop real time PCR assays for detection of all Phytoplasmas reported in citrus; optimize the assays so they may be performed as multiplex assays with assays for huanglongbing (HLB) and stubborn. 3) Conduct limited surveys in Riverside, San Diego, and Lindcove areas to determine incidence of Phytoplasma in citrus, assays for HLB and stubborn would be performed concurrently. 1b.Approach (from AD-416): Using DNA extracted from established planta cultures, the 16S rRNA region would be PCR amplified, cloned and sequenced. The sequences would be examined phylogenetically and the isolates of Phytoplasmas classified according to IRPCM guidelines. The sequence information would be utilized to develop Taqman-based real time PCR assays. The real time PCR assays would be optimized, and developed so that they may be performed in multiplex with real time PCR assays for citrus stubborn and huanglongbing. Limited surveys would be conducted in the areas of Riverside (which houses the genetic resources of the University of California, Riverside and the Repository), San Diego where the Asian citrus psyllid has recently been found, and in Lindcove area where the CCPP foundation block is maintained. 3.Progress Report: This is the final report for this research. This research relates to objective 3A of the parent project, "Recover citrus germplasm exposed to Huanglongbing (HLB) and citrus canker, and evaluate citrus relatives for tolerance or resistance to psyllids and/or HLB". The purpose of this research is to conduct a molecular characterization and determine the classification groups for the Phytoplasma found in citrus. We have established four California phytoplasma cultures in planta and have PCR amplified the 16S rDNA region and sequenced the amplified product to confirm the presence of phytoplasma. Additionally from the exotic citrus disease quarantine greenhouse in Beltsville, Maryland, we have PCR amplified and sequenced the 16S rDNA region of the witches’ broom disease phytoplasma originating from Oman and a witches’ broom on sweet orange originating from India. The sequence information from the phytoplasmas has been used to develop conventional polymerase chain reaction (PCR) assays and real time qPCR assays for generic detection of phytoplasmas and for specific phytoplasma. A more sensitive Taqman quantitative PCR (qPCR) assay has been developed for stubborn as part of this current project. Application of the qPCR assays have enabled detection of two different phytoplasmas from DNA extractions of samples collected from citrus and citrus relatives in South Florida. About 6,500 DNA extractions from Southern California have been assayed for phytoplasma, Candidatus Liberibacter species, and stubborn (stubborn disease), with no phytoplasma or Candidatus Liberibacter species being found. The overall impact of the accomplishments being the availability of reliable assays to detect phytoplasmas present in citrus and to differentiate them from stubborn disease and huanglongbing as the symptoms of all three diseases may be very similar.