Host-free biofilm culture of " Liberibacter asiaticus," the bacterium associated with Huanglongbing.

Inability to culture the phloem-restricted alpha-proteobacterium " Liberibacter asiaticus" (" L. asiaticus") or the closely related species (" Liberibacter americanus" and " Liberibacter africanus") that are associated with Huanglongbing (HLB) hampers the development of effective long-term control strategies for this devastating disease. Here we report successful establishment and long-term maintenance of host-free " L. asiaticus" cultures, with the bacterium growing within cultured biofilms derived from infected citrus tissue. The biofilms were grown in a newly designed growth medium under specific conditions. The initial biofilm-based culture has been successfully maintained for over two years and has undergone over a dozen subcultures. Multiple independent cultures have been established and maintained in a biofilm reactor system, opening the door to the development of pure culture of L. asiaticus and the use of genetics-based methods to understand and mitigate the spread of HLB