2011 Annual Report 1a.Objectives (from AD-416) Culture ZC Liberibacter bacterium and confirm its pathogenicity. 1b.Approach (from AD-416) The first step is to establish an in planta culture of Liberibacter. For in vitro culture, standard procedure for cultivation of fastidious prokaryotes will be followed. Liber A medium will be used as a starting point for cultivation. Growth of Liberibacter will be monitored by standard microbiological methods and by PCR detection. If Liberibacter are detected by PCR, various cultivation conditions and media compositions will be optimized for bacterial growth. Bacteria will be triple-cloned to obtain pure cultures. Transmission electron microscopy will be used for morphological characterization. 3.Progress Report This Reimbursable Agreement supports Objective 1.A of the parent project. The research objective was to study the newly found invasive bacterium, “Ca. L. solanacearum”, associated with potato zebra chip disease (ZCD). Endophytic bacteria in Zebra Chip Disease-affected potato tubers were partially characterized in this project. A total of 85 bacterial strains belonging to 17 bacterial genera were isolated by in vitro cultivation. ZCD tubers had significantly more culturable endophytic bacterial species than non-ZCD tubers. Paenibacillus sp., Microbacterium sp., Brachybacterium sp., Staphylococcus sp., Stenotrophomonas sp. and Klebsiella sp. were isolated only from ZCD potato tubers, whereas Bosea sp., Sphingopyxis sp., Sphingomonas sp.and Nocardia sp. were isolated only from non-ZCD potato tubers. Polymerase chian reaction (PCR) assays using specific primers further confirmed the presence of Microbacterium spp. in ZCD tubers and Bosea sp. in non-ZCD tubers. Scanning electron microscopy revealed bacteria with morphology of bacilli, coci, and pleomorphic shapes in the phloem tissue of potato tubers.