ARS | Publication request: Huanglongbing and psyllid cell cultures

Submitted to: Georgia Academy of Sciences Meeting
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 2, 2009
Publication Date: April 3, 2009
Citation: Hunter, W.B., Hert, M.M., Hall, D.G. 2009. Huanglongbing and psylid cell cultures [abstract]. Georgia Academy of Sciences Meeting. Paper No. 67.

Interpretive Summary: We successfully established cell cultures of the Asian citrus psyllid. Studies of the bacterial pathogens associated with huanglongbing, known as citrus greening disease, have long been impeded by the fact that the bacterium responsible for the disease has not yet been successfully cultured under artificial conditions. Reports that the disease pathogen is retained within the psyllid for 12 weeks suggested a new approach to culturing the pathogen: using psyllid cell cultures as the living medium in which to isolate and culture the bacterium. Several commercially available insect cell culture media were screened for suitability to culture cells from psyllid embryos. Successful psyllid cell cultures were obtained using a defined medium referred to as Hert-Hunter-70. This Asian citrus psyllid cell line provides a new tool for research on interactions between the bacteria and psyllid, and it may facilitate culturing not only the disease bacterium but also psyllid viruses that could be used as biological control agents.

Technical Abstract: We successfully established cell cultures of the Asian citrus psyllid, Diaphorina citri (Psyllidae: Hemiptera), DcHH-1. The cell culture also supported growth of Candidatus Liberibacter asiaticus. This bacterial pathogen is associated with Huanglongbing, known as citrus greening disease. Research on these bacteria have long been impeded by the fact that the bacterium Candidatus Liberibacter has not yet been successfully cultured under artificial conditions. Reports that Ca. Liberibacter asiaticus is retained within the psyllid for 12 weeks, suggested a new approach using psyllid cell cultures as the living medium to isolate and culture this bacterium. Successful psyllid cell cultures were obtained using a defined medium referred to as Hert-Hunter-70, HH70. Comparison of the HH70 media to others: [Ex-cell 405 (Sigma), Sf900 III SFM (GIBCO), Schneider¿s Insect Medium (Sigma), TNM-FH Insect Medium (Sigma), TC100 Insect Medium (Sigma), Shields and Sang M3 Insect Medium (Sigma), IPL-41 Insect Medium (Sigma)] demonstrated that media lacking or insufficient in: KCl, NaHCO3, NaH2PO4, Alanine, L-Cystine, p-Aminobenzioc acid, D-Biotin, D-Calcium pantothenate, Folic Acid, i-Inositol, Nicotic Acid, Pyridoxine, Riboflavin, Thiamine, Fumaric acid, alpha-Ketoglutaric acid, L-Malic acid and Succinic acid did not support psyllid cell growth. The psyllid cell line DcHH-1, grew attached to the substrate, and the suspended cells survived for several months. The doubling time of cells is approximately four days, with passage once every 8-10 days at a temperature of 25°C. The Diaphorina citri cell line, DcHH-1, provides a greatly needed research tool which will now be applied to studies of Ca. Liberibacter-psyllid cell interactions. These cell cultures also provide a highly controlled system for the study and propagation of psyllid viruses for use as biological control agents.