2008 Annual Report 1a.Objectives (from AD-416) The objectives are to culture the bacteria responsible for the Huanglongbing (HLB) disease of citrus and develop improved PCR-based detection assays. This research is described under CRIS objectives 1B, Developing a method to cultivate the HLB bacterium and 2B, Develop molecular techniques for identification of Liberabacter asiaticus, L. africanus, and L. americanus. 1b.Approach (from AD-416) For the first approach we will supplement various media designed for culturing fastidious bacteria with citrus extract. In a second approach we will determine the chemistry of citrus phloem and design special media based on metabolic pathways. 3.Progress Report The Huanglongbing (HLB) disease results in tree decline and death of citrus trees. Research into understanding and controlling the highly destructive disease has been hampered by the inability to culture the causal bacterium. Diagnosis of the disease has focused on symptoms and the presence of a group of bacteria known as Liberibacter, which occur in association with the disease. Three different species, L. asiaticus, L. africanus, and L. americanus, have been identified by sequencing a single gene region known as 16s rDNA. Polymerase chain reaction assays have been developed to detect the bacterium in citrus using this locus. Using a newly designed medium, we have obtained limited cultivation of a bacterium identified as L. asiaticus. A small amount of pure culture has allowed us to initiate pathogenicity tests using seedlings in a growth chamber and DNA has been submitted Los Alamos National Laboratories for sequencing the genome. The inoculated orange seedlings have shown some mottling symptoms and the bacterium has been re-isolated. Improvements are being made in our medium to improve the growth of the bacterium and pathogenicity tests are planned under field conditions to complete Koch’s postulates. This research falls within ARS National Program 303, Plant Diseases. Specifically addressing identification, characterization, and detection of foreign and newly emerging pathogens.