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SYBR Green real-time quantitative PCR for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in field samples from New Zealand

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Abstract

Diseases of solanaceous crops caused by the phloem-limited bacterium ‘Candidatus Liberibacter solanacearum’ (Lso), vectored by the tomato potato psyllid Bactericera cockerelli, pose a major economic threat to crop production. Lso is yet to be cultured and, therefore, effective control strategies depend heavily on the early detection of the pathogen via polymerase chain reaction (PCR) assays. In this study, two new assays for the detection of Lso in New Zealand field samples were developed, and compared with previously available assays. Firstly, a single-tube semi-nested gel-based PCR assay was developed for the genus-specific detection of liberibacter species, and shown to provide increased sensitivity over standard and nested PCR. Secondly, a single-tube semi-nested SYBR Green real-time PCR (qPCR) assay was developed for the specific detection of Lso in field samples from New Zealand, with a limit of detection of five copies of the target gene per reaction. Semi-nested qPCR showed similar sensitivity compared with TaqMan qPCR with the primer-probe combination LsoF-HLBpr and was 10- to 50-fold more sensitive than the conventional PCR assays tested. Quantification of titre in Lso-affected tubers by SYBR Green qPCR revealed a positive relationship between pathogen titre and the discolouration of fried tuber slices, a symptom indicative of Lso infection. Quantification of Lso in field samples of potato and tomato also revealed many samples with titres below the limit of detection of conventional PCR. The observation of low-titre samples demonstrated the utility of SYBR Green qPCR for detection of Lso, as in addition to increased sensitivity melt-curve analysis enables confirmation of qPCR data by identifying false positive results.

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Acknowledgments

We thank Dr Lia Liefting (MAF Biosecurity, New Zealand), Dr Fiona Constable (Department of Primary Industries, Victoria, Australia) and Dr Elena Gonella (University of Turin, Italy) for the provision of DNA samples from infected plant material; Dr Stephen Ogden (Potatoes New Zealand Psyllid Coordinator) for supplying NZ business impact data; Pauline Cooper (The New Zealand Institute for Plant & Food Research Limited) for the provision of potato tissue culture plantlets, and Ruth Butler (The New Zealand Institute for Plant & Food Research Limited) for valuable assistance with the manuscript. This work was supported by a Capability Grant Fund from The New Zealand Institute of Plant & Food Research Limited and the Ministry of Agriculture and Fisheries Sustainable Farming Fund (Sustainable Tomato/Potato Psyllid Management 09/143).

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Correspondence to I. A. W. Scott.

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Beard, S.S., Pitman, A.R., Kraberger, S. et al. SYBR Green real-time quantitative PCR for the specific detection and quantification of ‘Candidatus Liberibacter solanacearum’ in field samples from New Zealand. Eur J Plant Pathol 136, 203–215 (2013). https://doi.org/10.1007/s10658-012-0156-5

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