Submitted to: International Research Conference on Huanglongbing
Publication Type: Proceedings
Publication Acceptance Date: December 2, 2010
Publication Date: January 1, 2011
Citation: Ammar, E., Shatters, R.G., Hall, D.G. 2011. Localization of Candidatus Liberibacter asiaticus in dissected organs of its psyllid vector Diaphorina citri using fluorescent in situ hybridization and quantitative PCR. 2nd International Research Conference on Huanglongbing, Jan. 10-14, 2011, Orlando, Florida. Paper No. 3-3. Interpretive Summary: Vector interactions of huanglongbing (HLB) disease with its psyllid vectors, particularly at the organ and cellular levels, are poorly understood. We used fluorescent in situ hybridization (FISH) and quantitative PCR for the localization of Candidatus Liberibacter asiaticus, associated with HLB, in its vector the Asian citrus psyllid Diaphorina citri.
Technical Abstract: Vector interactions of huanglongbing (HLB) disease with its psyllid vectors, particularly at the organ and cellular levels, are poorly understood. We used fluorescent in situ hybridization (FISH) and quantitative PCR (Q-PCR) for the localization of Candidatus Liberibacter asiaticus (CLas), associated with HLB, in its psyllid vector Diaphorina citri. Several FISH protocols have been tested on hemolymph smears and dissected psyllid organs and on leaf sections from HLB-infected citrus plants as positive controls. CLas was detected in the hemolymph, filter chamber and midgut of D. citri collected from field HLB-infected citrus trees, as well as in the phloem of infected leaves, but not in healthy control psyllids or leaves. Additionally, Q-PCR detected CLas in dissected organs of individual D. citri adults collected from HLB-infected citrus trees. The proportion of infected salivary glands (47 percent) was significantly lower than those of the alimentary canal (72 percent) or other body parts (79 percent). Interestingly, the relative titer of CLas, compared to psyllid genomic DNA in each sample, was significantly higher in both the salivary gland and alimentary canal compared to that in the rest of the insect body. These results provide the first molecular confirmation of CLas in the hemolymph, alimentary canal and salivary glands of D. citri. They also strongly suggest that the salivary glands constitute a major transmission barrier to CLas in the psyllid vector, and that CLas may replicate or accumulate in both the alimentary canal and salivary gland of D. citri.