Studying Liberibacter crescens to learn more about the causal agent of huanglongbing

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What is the problem?

Studying how a pathogen interacts with its plant host and its insect vector is of great importance for understanding disease development and transmission in order to prevent and control plant diseases. Many of these studies are performed under greenhouse and field conditions; however, such investigations also require laboratory studies of the organism in 'pure culture', in the absence of other microorganisms, and under controlled conditions. Without these controlled experiments, it is impossible to study a microbe’s growth requirements or its pathogenic weapons, also known as virulence factors. To do this, it requires mimicking the natural environments in which each specific bacterium lives and reproduces in the lab.

The main hurdle in studying the huanglongbing bacterium, "Candidatus Liberibacter asiaticus" (CLas), and its interactions with its host citrus species and insect vector, the Asian citrus psyllid, is the lack of a medium that supports its growth in pure culture. CLas lives in citrus phloem and has adapted very well to this environment. Because of this, it cannot produce most metabolites required for its own development. Thus, CLas must acquire them from outside--in the same way plants need water and nutrients provided to them by humans. Despite multiple attempts to recreate a medium that replicates the conditions in the phloem or in specific psyllid tissues, the complexity of CLas niches, as well as our limited understanding of CLas metabolic pathways, has prevented being able to culture this pathogen.

What is the technique?

Worldwide efforts are being made by researchers to grow CLas, including the use of surrogates of bacteria that are closely related to CLas. The best model to date is Liberibacter crescens (Figure 1), the only culturable member of the group of bacteria to which CLas belongs. This bacterium, first isolated in 1995 from a diseased mountain papaya tree in Puerto Rico, is not pathogenic and is not transmitted by any known vector. Thus, no quarantine measures are needed to work with it in HLB-free regions. L. crescens shares 94% of the genetic information (DNA) of CLas, and the similarity of the genes required for important metabolic pathways can help identify which factors may affect the inability to culture CLas. In addition, L. crescens can be genetically modified to study the function of genes shared with CLas, or to produce CLas proteins. Because of its similarity, L. crescens is the best bacterial model for studying huanglongbing and other diseases caused by Liberibacter species, like zebra chip disease of potato.

Despite its being the best CLas surrogate, L. crescens is still challenging to grow. In our studies, we greatly improved L. crescens’ growth rate and survival in the laboratory by altering multiple culture conditions and developing new growth medium formulations. Furthermore, we showed that L. crescens consumes organic acids rapidly, creating poisonously alkaline conditions in the growth medium that impede the ability of L. crescens to reproduce, and therefore its ability to be cultured (Figure 2). In addition, our results were used to build a mathematical model of L. crescens growth, based on bacterial stress and adaptation to deleterious conditions. This will help better understand L. crescens growth and predict conditions that will help in designing new growth medium as well as a first approach to understand why CLas can’t be cultured. This model fits the L. crescens culture data, but more importantly was used to make predictions that have been confirmed experimentally, e.g., culturability loss is reduced when the pH is corrected by organic acid addition the day the culture reaches pH 8.5 (day 5 of growth in our conditions), but not a day after showing that alkaline stress is crucial for L. crescens culturability.

Who is working on the project?

Dr. Marta Sena-Vélez, Dr. Kathryn Jones, Sean Holland and Darryl Trickey at Florida State University, Department of Biology, and Drs. Dean Gabriel and Mukesh Jain at the University of Florida, Department of Plant Pathology, are working on culturing L. crescens and CLas. Drs. Manu Aggarwal and Nick Cogan at FSU, Department of Mathematics, are working on mathematical modeling of L. crescens growth and metabolism.

What are the challenges and opportunities?

Similarity in core metabolic pathways for carbon and nitrogen utilization between L. crescens and CLas means that work on culture requirements of L. crescens provides valuable information for the development of a medium that can be used to grow CLas. Our finding that L. crescens creates alkaline conditions in its medium (Figure 2 red line), such that it poisons itself, is unusual for a bacterium. Determining if such alkali production plays a role in the failure of CLas to grow in culture is one avenue for investigation opened up by our research on L. crescens. Further approaches include investigation of other biochemical pathways related to the utilization of different nutrient sources in both L. crescens and CLas. Improved growth of L. crescens in the laboratory will make it easier to use this model to study CLas proteins important for communication with the environment and gene regulation networks and to better comprehend CLas behavior in both nature and the laboratory. An improved understanding of the basic biology of CLas will help us understand how to fight it. Use of L. crescens as a culturable surrogate for CLas has allowed us to gain a foothold in understanding Liberibacter biology and will permit the development of new tools to further understand and control the devastating huanglongbing disease of citrus.

Funding source:
This work was funded by the National Institute of Food and Agriculture,
U.S. Department of Agriculture, under award number 2014-67013-21579 to K.M.J., USDA NIFA SCRI Citrus Disease Research and Extension (CDRE)
award 2016-70016-24844 to D.H.G. and subaward UFDSP00011165 to K.M.J