Title: Low-level detection of Candidatus Liberibacter solanacearum in extracted Bactericera cockerelli (Hemiptera: Triozidae) DNA by 454 pyrosequencing Authors
|Arp, Alex -|
|Chapman, Rebekah -|
|Bextine, Blake -|
Submitted to: Environmental Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 19, 2013
Publication Date: October 20, 2013
Citation: Arp, A., Chapman, R., Crosslin, J., Bextine, B. 2013. Low-level detection of Candidatus Liberibacter solanacearum in extracted Bactericera cockerelli (Hemiptera: Triozidae) DNA by 454 pyrosequencing. Environmental Entomology. 42:868-873. Interpretive Summary: Potato zebra chip disease (ZC) is caused by a bacterium transmitted to potatoes by the potato psyllid. The disease has caused severe economic damage in Mexico, Central America, New Zealand, the south-central United States. Since the ZC bacterium, Candidatus Liberibacter solanacearum, is transmitted by the potato psyllid, testing of infected psyllids is imperative. Recent evidence suggests that the level of liberibacter in psyllids can vary widely and even be undetectible using standard molecular tests. The work described here shows that extremely low levels of bacterial infection in psyllids is indeed possible and can be detected using the new generation DNA sequencing techniques.
Technical Abstract: Accurate detection and quantification of Candidatus Liberibacter solanacearum, the putative causal agent of zebra chip disease of potato (Solanum tuberosum), in the potato psyllid, Bactericera cockerelli, has become necessary to better understand the biology of the disease cycle. Studies on the transmission efficiency potato psyllids have shown inconsistencies with field surveys. There have also been reports of laboratory colonies inexplicably losing and regaining Candidatus Liberibacter solanacearum infection. Until now, DNA primers were used to detect Candidatus Liberibacter solanacearum in potato psyllid tissue using conventional polymerase chain reaction and gel electrophoresis or by real-time quantitative PCR. In this study, Candidatus Liberibacter solanacearum was detected using bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) at levels identifiable by PCR, and low levels, including samples with only one cell of Candidatus Liberibacter solanacearum. Potato psyllids with <300 pyrosequencing reads did not show positive using conventional PCR. These results explain why it is possible for laboratory colonies to seemingly lose and regain Candidatus Liberibacter solanacearum infection and possibly why some populations of potato psyllids that do not test positive for Candidatus Liberibacter solanacearum still occur in potato fields with symptoms of zebra chip .