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.

With the current advancements in DNA sequencing technology, the limiting factor in long-read metagenomic assemblies is now the quantity and quality of input DNA. Although these requirements can be met through the use of axenic bacterial cultures or large amounts of biological material, insect systems that contain unculturable bacteria or that contain a low amount of available DNA cannot fully utilize the benefits of third-generation sequencing. The citrus greening disease insect vector Diaphorina citri is an example that exhibits both of these limitations. Although endosymbiont genomes have mostly been closed after the short-read sequencing of amplified template DNA, creating de novo long-read genomes from the unamplified DNA of an insect population may benefit communities using bioinformatics to study insect pathosystems. Here all four genomes of the infected D. citri microbiome were sequenced to closure using unamplified template DNA and two long-read sequencing technologies. Avoiding amplification bias and using long reads to assemble the bacterial genomes allowed for the circularization of the Wolbachia endosymbiont of Diaphorina citri for the first time and paralleled the annotation context of all four reference genomes without utilizing a traditional hybrid assembly. The strategies detailed here are suitable for the sequencing of other insect systems for which the input DNA, time, and cost are an issue.

Background Liberibacter crescens is the closest cultured relative of four important uncultured crop pathogens. Candidatus. L. asiaticus, L. americanus, L. africanus cause citrus greening disease, while Ca. L. solanacearum causes potato Zebra chip disease. None of the pathogens grows in axenic culture. L. crescens grows in three media: a BM-7, a serum-free Hi® Grace’s Insect Medium (Hi-GI), and a chemically-defined medium called M15. To date, no optimal growth parameters of the model species L. crescens have been reported. Studying the main growth parameters of L. crescens in axenic culture will give us insights into the lifestyle of the Ca. Liberibacter pathogens. Results The evaluation of the growth parameters—pH, aeration, temperature, and buffering capacity—reflects the optimal living conditions of L. crescens. These variables revealed that L. crescens is an aerobic, neutrophilic bacterium, that grows optimally in broth in a pH range of 5.8 to 6.8, in a fully oxygenated environment (250 rpm), at 28 °C, and with monosodium phosphate (10 mM or 11.69 mM) as the preferred buffer for growth. The increase of pH in the external media likely results from the deamination activity within the cell, with the concomitant over-production of ammonium in the external medium. Conclusion L. crescens and the Ca. Liberibacter pathogens are metabolically similar and grow in similar environments—the phloem and the gut of their insect vectors. The evaluation of the growth parameters of L. crescens reveals the lifestyle of Liberibacter, elucidating ammonium and phosphate as essential molecules for colonization within the hosts. Ammonium is the main driver of pH modulation by active deamination of amino acids in the L. crescens amino acid rich media. In plants, excess ammonium induces ionic imbalances, oxidative stress, and pH disturbances across cell membranes, causing stunted root and shoot growth and chlorosis—the common symptoms of HLB-disease. Phosphate, which is also present in Ca. L. asiaticus hosts, is the preferred buffer for the growth of L. crescens. The interplay between ammonium, sucrose, potassium (K+), phosphate, nitrate (NO3−), light and other photosynthates might lead to develop better strategies for disease management.

Citrus greening disease is now completely endemic to Florida citrus trees, having spread rapidly across all counties in the past 20 years and causing devastating economic losses. The disease’s etiological agent is the phloem-restricted bacterium, Candidatus Liberibacter asiaticus (CLas). Liberibacter crescens, the closest culturable relative, was discovered to prefer citrate as its most effective carbon and energy source. Citrus plants load free-citrate in response to phosphorus deficiency. In Florida’s calcareous soils, supplemental phosphate fertilization is very low due to the assumption that it is readily available for plants through the soil. It is likely that citrus trees are loading citrate to mine phosphorus from the soil, which could inadvertently exacerbate CLas infection. In greenhouse experiments, foliar sprayed phosphate is taken up by the plants, resulting in decreased free-citrate levels and changes in fumaric and succinic acids in the phloem, in addition to delaying titer detection of CLas. Our field experiments - where mature trees have a well-established HLB infection that’s maintained by infected psyllids – exhibited lower CLas titer for some of the phosphate treatments across time.