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Title: Development of primers and probes for genus and species specific detection of 'Candidatus Liberibacter species' by real-time PCR

Author
item ANANTHAKRISHNAN, G - University Of Florida
item CHOUDHARY, N - University Of Florida
item ROY, A - University Of Florida
item SENGODA, V - Yakima City Extension Service
item Postnikova, Elena
item Hartung, John
item Stone, Andrew
item Damsteegt, Vernon
item Schneider, William
item Munyaneza, Joseph - Joe
item BRLANSKY, R - University Of Florida

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2013
Publication Date: 3/25/2013
Citation: Ananthakrishnan, G., Choudhary, N., Roy, A., Sengoda, V.G., Postnikova, E.N., Hartung, J.S., Stone, A.L., Damsteegt, V.D., Schneider, W.L., Munyaneza, J.E., Brlansky, R.H. 2013. Development of primers and probes for genus and species specific detection of 'Candidatus Liberibacter species' by real-time PCR. Plant Disease. 97:1235-1243.

Interpretive Summary: Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease sweet orange and other citrus trees. Three forms of the disease are currently known and are associated with three different but closely related species of bacteria. The symptoms of the diseases are very similar in infected trees, but the insect vectors and disease progress potentials are different, so accurate diagnosis is important. These bacteria can’t be cultured in the laboratory, so diagnosis and detection of the pathogens is done by a laboratory technique called qPCR. There is a fourth species of bacterium closely related to the species infecting citrus that infects potatoes and tomatoes. Each of these bacteria can be detected by separate q-PCR tests, but separate tests for each pathogen are time consuming and expensive. We have developed, for the first time, a single q-PCR assay that will detect and identify all four pathogens, if present, in a single sample. This new set of assays will simplify diagnostic testing for this group of pathogens. It will be useful to scientists and diagnosticians working with this group of pathogens, and may be used either as a supplement to or a replacement of current methods.

Technical Abstract: Huanglongbing (HLB), also known as citrus greening, is currently the most devastating disease 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 12 (qPCR). The most widely used assays use primers and probes based on the 16S ribosomal RNA 13 (rRNA) gene. Recent molecular analyses of ‘Candidatus Liberibacter species’ and other bacteria suggest that the rpoB gene (encoding the ß-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 africanus’ and ‘Ca. Liberibacter americanus’). 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 fully 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.