|Stone, Andrew - Andy|
Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/13/2008
Publication Date: 5/1/2009
Citation: Sechler, A.J., Schuenzel, E., Cooke, P.H., Donnua, S., Thaveechai, N., Postnikova, E.N., Stone, A.L., Schneider, W.L., Damsteegt, V.D., Schaad, N.W. 2009. Cultivation of Candidatus Liberibacter asiaticus and Ca. L. americanus associated with Huanglongbing. Phytopathology. 99:480-486. Interpretive Summary: Huanglongbing (HLB) disease of citrus, also known as “citrus greening,” is considered the most destructive disease of citrus species in the world and currently threatens the existence of the citrus industry in many countries. The disease causes a rapid tree decline, characterized by yellowed shoots with blotchy mottled leaves, small, poor-quality, lopsided fruits with color inversion, and aborted seeds. The suspected causal agents of HLB are bacteria from the Candidatus Liberibacter genus, Ca. L. africanus, Ca. L. asiaticus, and Ca. L. americanus. The causal species of the bacteria depends on the climate and country where the disease was found. None of the three described species of bacteria can be cultivated on artificial media nor have they been shown to cause HLB-like symptoms when inoculated into citrus. We designed and tested a new artificial medium, Liber A, that allows for the cultivation of all three species of Liberibacter on it. Two strains of Ca. L. asiaticus and one of Ca. L. Americanus grown on Liber A medium were pathogenic on citrus and could be isolated from non-inoculated tissues of inoculated trees and seedlings nine and two months later respectively. Each isolated strain was shown to be the same organism by molecular assays. This is the first report of the cultivation and pathogenicity of Ca. L. asiaticus and Ca. L americanus associated with symptoms of HLB.
Technical Abstract: A new medium designated Liber A has been designed and used to successfully cultivate all three Candidatus Liberibacter species, the suspect causative agents of Huanglongbing (HLB) in citrus. The medium containing citrus vein extract and a growth factor sustained growth of Ca. Liberibacter species for four or five single colony transfers before viability declined. Colonies, positive for Ca. L. asiaticus by a 16s-based rDNA real-time polymerase chain reaction (RT-PCR) assay and sequencing, were irregular-shaped, convex and 0.1 to 0.3 mm after 3 to 4 days. Suspect Ca. L. asiaticus and Ca. L. americanus cells were observed in infected tissue and on agar culture by scanning electron microscopy. The cells were ovoid to rod shaped 0.3 to 0.4 x 0.5 to 2.0 µm with numerous fimbriae-like appendages. Two strains of Ca. L. asiaticus and one of Ca. L americanus grown on Liber A medium were pathogenic on citrus and could be isolated from non-inoculated tissues of inoculated trees and seedlings nine and two months later respectively. The identity was confirmed by RT-PCR and 16s rDNA sequencing. This is the first report of the cultivation and pathogenicity of Ca. L. asiaticus and Ca. L. americanus associated with symptoms of HLB.