Quantification of Candidatus Liberibacter Asiaticus In Plant Extracts – From Ct to Biologically Meaningful Units

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 nonsensical 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  10⁰ 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 4) 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.