Submitted to: Molecular and Cellular Probes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/14/2011
Publication Date: 4/1/2012
Citation: Morgan, J.K., Zhou, L., Li, W., Shatters, R.G., Keremane, M.L., Duan, Y. 2012. Improved real-time PCR detection of Candidatus Liberibacter asiaticus from citrus and psyllid hosts by targeting the intragenic tandem repeats of its prophage genes. Molecular and Cellular Probes. 26:90-98. Interpretive Summary: Citrus huanglongbing (HLB) is a devastating disease of citrus worldwide. The HLB bacteria reside in the sieve cells of their host plants at variable bacterial titers. Detection of these bacteria has proven difficult because of their low titer. This report characterizes the first use of real-time PCR primers targeting multiple tandem repeats within two prophage genes of the Candidatus Liberibacter asiaticus (Las) genome using both SYBR Green 1 (LJ900fr) and TaqMan® (LJ900fpr) methods. These methods dramatically enhance the detection of the Las bacterium. Because the target concentration within each cell increased when probing for the repeat region, the level of detection was also improved, which translated into =100 fold increase (LJ900fr) in the detection level relative to the present real-time standard for Ca. L. asiaticus using the 16S rDNA-based HLBaspr. Meanwhile, these real-time PCR methods with LJ900fr and LJ900fpr apply different chemistries and thereby have different sensitivities, which provide users a flexible choice based on their objectives. The increased sensitivity provided by these methods allows detection of Ca. L. asiaticus from some symptomatic HLB samples that are negative by alternate detection methods. However, the detection of the extreme low titer by LJ900fr (SYBR Green 1) also revealed that the presence of the Las bacterium may not equal the presence of HLB disease because many of these Las-positive plants with an extremely low bacterial titer did not develop typical HLB disease for several years. These results suggest the presence of weak or non-pathogenic forms of Ca. L. asiaticus.
Technical Abstract: Candidatus Liberibacter asiaticus (Las) is the most prevalent species of Liberibacter associated with citrus huanglongbing (HLB) worldwide. Residing in phloem sieve cells of host plants and vectored by the Asian citrus psyllid (Diaphorina citri), this fastidious bacterium lives as a pathogen or symbiont. Uneven distribution coupled with a dramatic variation of the bacterial titers in their hosts emphasizes the need for sensitive and reliable detection. Here we developed a real-time PCR assay using SYBR Green 1 (LJ900fr) and TaqMan® (LJ900fpr) protocols with primers and probe targeting nearly identical tandem repeats of 132bp within two Las prophage genes, hyvI and hyvII. Because the copy number of the tandem repeats is higher than other targets per bacterial genome, these methods significantly improved the detection capacity of the HLB bacterium. In comparable samples relative to 16S HLBaspr real-time PCR, these new methods reduced the relative detectable threshold by approximate 9 and 3 cycles for LJ900fr and LJ900fpr, respectively. From HLB samples with an extremely low titer of Las bacterium, both LJ900fr and LJ900fpr detected Las from otherwise non-detectable samples by HLBaspr. The discrepancy in Ct values between prophage- and 16S rDNA-based assays suggests variable numbers and genetic variations of the tandem repeats of hyvI/II genes and the presence of weak or nonpathogenic forms among the Las isolates.