Development of Chemically Defined Media Reveals Citrate as Preferred Carbon Source for Liberibacter Growth

is the closest cultured relative of four important uncultured crop pathogens. L. asiaticus, L. americanus, and L. africanus are causal agents of citrus greening disease, otherwise known as huanglongling (HLB). L. solanacearum is responsible for potato Zebra chip disease. Cultures of grow slowly on BM-7 complex medium, while attempts to culture the Liberibacter pathogens in BM-7 have failed. Developing a defined medium for the growth of will be useful in the study of metabolism and will improve the prospects for culturing the . Liberibacter pathogens. Here, M15 medium is presented and described as the first chemically defined medium for the growth of cultures that approaches the growth rates obtained with BM-7. The development of M15 was a four step process including: (1) the identification of Hi-Graces Insect medium (Hi-GI) as an essential, yet undefined component in BM-7, for the growth of , (2) metabolomic reconstruction of Hi-GI to create a defined medium for the growth of cultures, and (3) the discovery of citrate as the preferred carbon and energy source for growth. The composition of M15 medium includes inorganic salts as in the Hi-GI formula, amino acids derived from the metabolomic analyses of Hi-GI, and a 10-fold increase in vitamins compared to the Hi-GI formula, with exception choline chloride, which was increased 5000-fold in M15. Since genome comparisons of and the . Liberibacter pathogens show that they are very similar metabolically. Thus, these results imply citrate and other TCA cycle intermediates are main energy sources for these pathogens in their insect and plant hosts. Thus, strategies to reduce citrate levels in the habitats of these pathogens may be effective in reducing Liberibacter pathogen populations thereby reducing symptoms in the plant host.