Publications - Faculty - Recently Submitted Faculty Publications 2010
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Brlansky - Plant Disease
Development of primers and probes for genus and species specific detection of ‘Candidatus Liberibacter species’ by real-time PCR
G. Ananthakrishnan, N. Choudhary and Avijit Roy, University of Florida, CREC, Lake Alfred, FL 33850; E. Postnikova, USDA-ARS, FDWSRU, Fort Detrick, MD 21702; J. S. Hartung, USDA-ARS, MPPL, Beltsville, MD 20705; A. L. Stone, V. D. Damsteegt and W.L. Schneider, USDA-ARS, FDWSRU, Fort Detrick, MD 21702; and R. H. Brlansky, University of Florida, CREC, Lake Alfred, FL 33850
Abstract Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease of Citrus. Three types of the disease are currently known and are associated with three different ‘Candidatus Liberibacter species’. When HLB-symptomatic trees are tested, one of three species of the Candidatus Liberibacters is normally detected by conventional or real-time PCR (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA (rRNA) gene. Recent molecular analyses of ‘Candidatus Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the β-subunit of RNA polymerase), is useful for bacterial identification. We report here the design of a pair of genus specific primers and a hybridization probe corresponding to the rpoB region and their application for the detection of all three known citrus Liberibacter species (‘Ca. Liberibacter asiaticus’, ‘Ca. Liberibacter americanus’ and ‘Ca. Liberibacter africanus’). In addition, species specific primers and probes based on the rplJ/rplK genes were designed and used for detection at the species level in a multiplexed format. Both the genus and species specific assays were validated in SYBR Green I and TaqMan formats. These one step qPCR diagnostic methods are useful for the detection of all species of citrus-infecting Liberibacter. In addition, the genus specific primers and probe successfully detected ‘Candidatus Liberibacter solanacearum’ associated with the potato Zebra Chip disease.
