2012 Annual Report 1a.Objectives (from AD-416): Culture ZC Liberibacter bacterium and confirm its pathogenicity 1b.Approach (from AD-416): The initial goal 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 is 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. Bacteriophages will also be explored for their relationships to pathogenicity. 3.Progress Report: This research is in support of Objective 1.A., of the in house project, “Candidatus Liberibacter solanacearum”, the putative pathogen of zebra chip disease of potato, was maintained and enriched using tomato plant as a host or “medium” for in planta culture. Techniques were developed for quick propagation and growth of infected tomato plants under greenhouse conditions. The plants were grown in potted soil or nutrient solution (hydroponic culture). Examination by transmission electron microscopy revealed that plants grown using both techniques accumulated walled bacteria, presumably “Ca. L. solanacearum”, in phloem tissue. The hydroponic solution culture system was used to evaluate how nutrients affected growth of “Ca. L. solanacearum”. Preliminary results showed that lack of nutrient(s) did not significantly affect accumulation of “Ca. L. solanacearum” despite reduced vegetative growth of plants.