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

Authors: Zheng, Desen; Armstrong, Cheryl; YAO, WEI - University Of Florida; WU, BO - Clemson University; LUO, WEIQI - North Carolina State University; POWELL, CHARLES - University Of Florida; Hunter, Wayne; LUO, FENG - Clemson University; GABRIEL, DEAN - University Of Florida; Duan, Ping

Submitted to: Horticulture Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/1/2024
Publication Date: 1/10/2024
Citation: Zheng, D., Armstrong, C.M., Yao, W., Wu, B., Luo, W., Powell, C., Hunter, W.B., Luo, F., Gabriel, D., Duan, Y. Towards the completion of Koch’s postulates for the citrus huanglongbing bacterium, Candidatus Liberibacter asiaticus. Horticulture Research. 2024. https://doi.org/10.1093/hr/uhae011.
DOI: https://doi.org/10.1093/hr/uhae011

Interpretive Summary: Huanglongbing (HLB), also known as citrus greeting, is the most devastating disease of citrus worldwide. The causal agents of HLB are intracellular bacterial pathogens, and one of them named as Candidatus Liberibacter asiaticus (Las). Due to the small genome and fastidious nature, culturing Las in vitro has proven to be extremely challenging. Previous works on Las culture have added to the body of knowledge, although no published culture method has been widely adopted. In this study, we optimized growth conditions and developed a semi-selective media based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the Las growth (LG) liquid medium with ca. 100 to 1000-fold increase and an estimated 1,000,000-10,000,000 Las cells/ml being obtained. We then inoculated the co-cultured Las back to citrus by first feeding them to psyllids and subsequently incubating the Las-fed psyllids on citrus seedlings for 2-3 weeks. Using these methods, we were able to demonstrate that the cultured Las caused typical HLB. The developed system for Las co-culture and inoculation back to citrus provides simple but semi-selective medium with a defined medium composition, and a basic foundation for improvement toward axenic culture and genetic modification of Las as well as a high-throughput screening for antimicrobial compounds.

Technical 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.100-1000-fold increase, which peaked after 4-6 weeks and were estimated to contain 106-107 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.