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.C.(identification of regulatory molecules of plant pathogens) of the in-house project. Concentration of “Candidatus Liberibacter solanacearum”, the pathogen associated with zebra chip disease of potato, is low in plant tissue, making standard microbiological research methods impractical. The problem was addressed through a leaf culture technique that increased bacterial concentration. Infected tomato leaves were planted in potted soil under greenhouse conditions or in sterile sand inside tissue culture tubes. An incubation time of 2 to 3 weeks increased bacterial concentration over one thousand fold, as measured by real-time polymerase chain reaction (PCR). Based on this observation, it was hypothesized that the leaf culture process may produce compounds that promote the bacterial growth or reduce antibacterial factors present in plant tissues. Further analyses to elucidate compounds/factors affecting bacterial growth may facilitate formulation of artificial media to culture the bacterium.