Title: Quantification of Candidatus Liberibacter asiaticus in plant extracts – from Ct to biologically meaningful units Authors
Submitted to: Proceedings American Society of Horticultural Sciences
Publication Type: Abstract Only
Publication Acceptance Date: April 9, 2012
Publication Date: N/A
Interpretive Summary: Huanglongbing (HLB) disease is having a devastating impact on the Florida citrus industry; the disease has recently been confirmed present in California, and poses a serious threat to that industry. HLB diagnosis is difficult because disease symptoms are easily confused with other disorders, especially micronutrient deficiencies. Candidatus Liberibacter asiaticus (CLas) is the bacterial plant pathogen believed to cause HLB and the presence of CLas in citrus is used to confirm HLB. Quantitative real time polymerase chain reaction is used to detect CLas cells for confirmation of CLas infection in citrus trees with suspect HLB symptoms. We were interested in determining how results of quantiative chain reaction could be used to quantify CLas cells rather than merely confirm infection. Numerous factors can affect the detection of CLas DNA extracted from citrus leaves and limits of detection for the assay are ambiguous. We provide evidence that using quantiative polymerase chain reaction it is possible to detect single CLas bacterial cells and that the working range for the assay is from 1 to ten million cells. Our results suggest that it is not possible to develop an assay with greater sensitivity than quantitative polymerase chain reaction for CLas detection. We also determined that the upper limit of CLas titer in plant tissues is in the range of ten million to 100 million CLas cells per gram of citrus leaf tissue. We used our standard curve to calculate the number of CLas cells per gram tissue for 20,000 field samples collected by commercial scouts. Analysis of the data reveals that: 1) the majority of samples were CLas-negative; 2) CLas-infected, but HLB-asymptomatic leaves have CLas titers of ten to ten thousand CLas cells per gram tissue; 3) the titer of CLas in HLB-symptomatic tissues is one million to ten million cells per gram tissue; and 4) no samples exceed one hundred million CLas cells per gram tissue. These results confirm the value of the standard curve method for conversion of quantitative polymerase chain reaction results to numbers of CLas cells and provide insights into the distribution of CLas titers in infected trees. This work has significant relevance for developing methods to screen new citrus germplasm for resistance to CLas infection and HLB disease.
Technical Abstract: Candidatus Liberibacter asiaticus (CLas) is a phloem-limited bacterium associated with the citrus disease Huanglongbing (HLB), regarded to be the most devastating disease of citrus. HLB has been confirmed in all citrus producing counties in Florida and threatens viability of the citrus industry. In the early stages of HLB, diagnosis is difficult because disease symptoms are easily confused with other disorders, especially micronutrient deficiencies. Initially, quantitative real time PCR (qPCR) of CLas 16S rDNA was used to confirm CLas infection in citrus trees with suspect HLB symptoms. Confirmatory testing is a dichotomous no/yes assay. Typically, qPCR results are expressed simply as crossing threshold (Ct) and values below an arbitrary Ct are interpreted as CLas positive. However, Ct values alone have no biological context and variability in qPCR protocols among laboratories makes the reliability of arbitrary Ct values for diagnostics questionable. Increasingly, qPCR is being used as a quantitative assay in experiments related to host –pathogen interactions and in attempts to quantify resistance to CLas. Conversion of Ct values to biological units (i.e. amount of CLas per mass tissue) provides biological context and allows for meaningful comparisons among laboratories. We developed a standard curve to convert Ct to biological units (CLas genomes) and have determined that the working range for qPCR quantification of CLas is 0 to 7 logs and that qPCR can detect a single copy of CLas 16S rDNA, indicating that it is not possible to develop an assay with greater sensitivity than qPCR for CLas detection. Conversion of CLas genomes to mass reveals that Ct values less than 18 are not meaningful because the amount of CLas would exceed > 1% of total DNA suggesting that the upper limit of CLas titer in plant tissues is 107 to 108 CLas genomes ·g-1. Using our standard curve to convert Ct values to CLas genomes ·g-1 tissue for 20,000 field samples collected by commercial scouts reveals that: 1) the majority of samples were CLas-negative (Ct > than 38.5); 2) CLas-infected, asymptomatic leaves have CLas titers of 102 to 104 genomes g·-1 tissue (Ct 38-30); 3) the titer of CLas in HLB-symptomatic tissues is 106 to 107 genomes ·g-1 tissues (Ct 24-19) and no samples exceed 108 CLas genomes g tissue (Ct < 18). These results confirm the value of the standard curve method for conversion of Ct to CLas genomes and provide insights into the distribution of CLas titers in infected trees.