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 in 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 research relates to objective 3A of the parent project. The purpose of this research is to conduct a molecular characterization and determine the classification groups for the Phytoplasma found in citrus. DNA extractions have been made from a number of apparent Phytoplasma diseases of citrus and citrus relatives from California and Florida. The 16S rRNA regions have been PCR amplified, and the products cloned and sequenced. Using this information, primers have been designed to enable detection of prokaryotes which may be present using qPCR and conventional PCR assays.