Publications - Faculty - Recently Submitted Faculty Publications 2008
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Wang - Plant Pathology
Quantitative real-time PCR assay for quantification of Candidatus Liberibacter asiaticusin citrus, periwinkle and psyllid
U.S. Sagaram, R.H. Brlansky, M. Rogers, L.L. Stelinski, C. Oswalt, J.S. Kim, and N. Wang
Abstract. In this study, a method using quantitative real-time PCR (qPCR) was developed for quantification of the Asiatic strain of citrus Huanglongbing (HLB) bacterium. Calibration curves were developed using plasmid templates consisting of 1409 bp and 703 bp DNA fragments of 16S rDNA and the rplKAJL-rpoBC (b-operon) region, respectively. Using this method, the actual detection limit falls between 20 and 200 copies, which is equivalent to between 2 × 104cells/g and 2 × 105 cells/g fresh tissue. Presence of plant DNA and other extracts did not have a considerable inhibitory effect on assay results. Candidatus Liberibacter asiaticus (Las) concentrations ranged from 7.84 ×103 to 2.14 × 107 CFU per µg of total DNA in citrus leaf midribs, roots, periwinkle leaves and psyllids. Our study also indicated that a minimum bacterial concentration is required for HLB symptom development in sweet orange. Quantification of the HLB bacterium in infected sweet oranges over a three-month span clearly showed a gradual increase in Las concentration in citrus over time.
