Towards the completion of Koch’s postulates for the citrus huanglongbing bacterium, Candidatus Liberibacter asiaticus

Abstract

Candidatus Liberibacter asiaticus (Las) is one of the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide. Due to the intracellular lifestyle and significant genome reduction, culturing Las in vitro has proven to be extremely challenging. In this study, we optimized growth conditions and developed a semi-selective medium based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the LG liquid medium with ca.100- to 1000-fold increase, which peaked after 4 to 6 weeks and were estimated to contain 10⁶ to 10⁷ cells/ml. The cultured Las bacteria remained in a dynamic state of growth for over 20 months and displayed limited growth in subcultures. The survival and growth of Las was confirmed by fluorescence in situ hybridization with Las-specific probes and expression of its metabolic genes. Growth of Las in the optimized medium relied on the presence of a helper bacterium, Stenotrophomonas maltophilia FLMAT-1 that is multi-drug resistant and dominant in the Las co-culture system. To recapitulate the disease, the co-cultured Las was inoculated back to citrus seedlings via psyllid feeding. Although the Las-positive rate of the fed psyllids and inoculated plants were relatively low, this is the first demonstration of partial fulfillment of Koch’s postulates with significant growth of Las in vitro and a successful inoculation of cultured Las back to psyllids and citrus plants that resulted in HLB symptoms. These results provide new insights into Las growth in vitro and a system for improvement towards axenic culture and anti-Las compound screening.

Acknowledgements

We are thankful to Dr. Nabil Killiny for providing technical guidance for the top feeding of psyllids. We would also like to thank Dr. David Hall, Dr. Robert Shatters, Kathy Moulton, Ellen Cochrane, and Anna Sara Hill for providing infected as well as uninfected psyllids, and Rachel Patterson, Christina Latza, and Spencer Marshall for technical support. Funding for this work was provided with the NIFA Award 2016-70016-24844 and 2016-10974 and USDA-ARS base funds.

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.

Author contributions

DZ, CA, and YD conceptualized this research. DZ, CA, WY, and WH investigated and validated the work.. DZ, CA, BW and WL provided formal analysis and data curation. DZ, CA, CP, DG and YD wrote and modified the manuscript. All authors involved in this study reviewed and edited the manuscript.

Data availability

The data that support the findings are available within the article and supplementary data.

Conflict of interest statement

The authors have no conflict of interest to declare.

Supplementary Data

Supplementary data is available at Horticulture Research online.

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