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United States Department of Agriculture

Agricultural Research Service

Research Project: IPM TECHNOLOGIES FOR SUBTROPICAL INSECT PESTS

Location: Subtropical Insects and Horticulture Research

Title: Establishment of Asian citrus psllid (Diaphorina citri) primary cultures

Authors
item Marutani-Hert, Mizuri -
item Hunter, Wayne
item Hall, David

Submitted to: In Vitro Cellular and Developmental Biology - Animals
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 4, 2009
Publication Date: August 8, 2009
Citation: Marutani-Hert, M., Hunter, W.B., Hall, D.G. 2009. Establishment of Asian citrus psyllid (Diaphorina citri) primary cultures. In Vitro Cellular and Developmental Biology-Animals. 45:317-320.

Interpretive Summary: A new cell line was developed from the Asian citrus psyllid (AsCP), Diaphorina citri (Hemiptera: Psyllidae), as a novel approach to culture the bacteria associated with huanglongbing disease (HLB), also known as citrus greening disease. The psyllid is a highly competent vector of the phloem-inhabiting bacterium Candidatus Liberibacter asiaticus associated with HLB. Severe losses in citrus are associated with HLB which causes reduced fruit yields, reduced juice quality and ultimately tree death. World-wide HLB has become a major limiting factor to the production of citrus. Efforts to manage HLB have been impeded by the fact that C. Liberibacter has not yet been cultured on artificial nutrient media. Since C. Liberibacter asiaticus is reported to replicate and survive in the psyllid, we developed a psyllid cell culture as the medium in which to culture the bacterium. We screened several commercially available insect cell culture media for suitability to culture cells from psyllid eggs, but ultimately we had to develop our own defined medium. We successfully established psyllid cell cultures of Diaphorina citri using a new media. Efforts are underway to inoculate these cells with C. Liberibacter asiaticus to determine if the bacteria will survive. If they survive, this will provide a much needed research tool to solve problems associated with HLB in citrus.

Technical Abstract: A new cell line was developed from the Asian citrus psyllid (AsCP), Diaphorina citri (Hemiptera: Psyllidae), as a novel approach to culture the bacteria associated with huanglongbing disease (HLB), also known as citrus greening disease. Methods to culture the phloem-inhabiting bacterium Candidatus Liberibacter asiaticus have been largely unsuccessful. Following reports that C. Liberibacter replicates within the psyllid host, we chose to develop a psyllid cell culture as the medium for culturing the bacterium. World-wide HLB has become a major limiting factor to the production of citrus. However, studies of HLB have been impeded by the fact that C. Liberibacter has not yet been cultured on artificial nutrient media. Several commercially available insect cell culture media were screened for suitability, but ultimately we developed our own defined medium to culture cells from AsCP embryos. Successful psyllid cell cultures, referred to as DcHH-1, were cultured with Hert-Hunter-70 medium, containing 10 percent FBS, and at a pH equal 6.5. The psyllid cells were attached to the substrate, fibroblast-like in morphology and are passed once every 8-10 days at a temperature of 25 degress C. Subsequent work is examining if C. Liberibacter asiaticus can survive and replicate within these cell cultures.

Last Modified: 9/3/2014
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