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

Abstract

Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus L. asiaticus, L. americanus, and L. africanus are causal agents of citrus greening disease, otherwise known as huanglongling (HLB). Candidatus L. solanacearum is responsible for potato Zebra chip disease. Cultures of L. crescens grow slowly on BM-7 complex medium, while attempts to culture the Ca. Liberibacter pathogens in BM-7 have failed. Developing a defined medium for the growth of L. crescens will be useful in the study of Liberibacter metabolism and will improve the prospects for culturing the Ca. Liberibacter pathogens. Here, M15 medium is presented and described as the first chemically defined medium for the growth of L. crescens 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 L. crescens, (2) metabolomic reconstruction of Hi-GI to create a defined medium for the growth of L. crescens cultures, and (3) the discovery of citrate as the preferred carbon and energy source for L. crescens 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 L. crescens and the Ca. 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 Ca. Liberibacter pathogen populations thereby reducing symptoms in the plant host.

Keywords: biochemical composition; chemically defined medium; culturing; metabolism.

FIGURE 1

The growth of Liberibacter crescens in BM-7 components. Cells were grown in Hi-GI and washed prior inoculation. Growth was monitored measuring optical density (OD₆₀₀) every 2 days for 8 days. The media containing TNM-FH and Hi-GI in the buffer system permit better growth of L. crescens compared to media with FBS in buffer or buffer alone. Growth in BM-7 was used as control.

FIGURE 2

Liberibacter crescens growth in different Grace’s Insect media. Cells were grown in Hi-GI and washed prior inoculation. Growth was monitored measuring optical density (OD₆₀₀) twice a day for ∼9 days. L. crescens grew significantly better in Hi-GI compared to Gibco-GI, Sigma-GI and Lab-GI media, and similar to BM-7 up to 116 h. (A) L. crescens growth in BM-7, Hi-GI (Hi-Media Labs), Gibco-GI (Gibco Technologies), and Sigma-GI (Sigma-Aldrich). (B) L. crescens growth in Grace’s Insect Medium prepared in the lab (Lab-GI) based on Hi-GI formula.

FIGURE 3

Liberibacter crescens growth in Hi-GI, M13, and M14 defined media. Cells were grown in Hi-GI and washed prior inoculation. Growth was monitored measuring optical density (OD₆₀₀) twice a day for ∼9 days. L. crescens grew similar in M13 and M14, but significantly less than Hi-GI. M14 medium contains all of the components of M13 except glucose, fructose, turanose, maltose, fumarate, α-ketoglutarate, malate, maleate, and succinate.

FIGURE 4

Liberibacter crescens growth tested in M14 medium with a range of citrate concentrations found in citrus phloem (200–6400 mg/L). Cells were grown in Hi-GI, and washed prior inoculation. Culture growth was monitored by measuring optical density (OD₆₀₀) twice a day for ∼11 days.

FIGURE 5

Carbohydrate source requirements for L. crescens growth with each of the sugars and organic acids at a concentration of 13 mM in M14 medium. A concentration of 13 mM is equivalent to 2.5 g/L citrate, the optimal level for growth of L. crescens. The sugars and organic acids present include α-ketoglutarate, malate, succinate, fumarate, citrate, glucose, fructose, turanose, and sucrose. M14 with 13 mM citrate is referred to as M15 medium.