Optimized Quantification of Unculturable Candidatus Liberibacter Spp. in Host Plants Using Real-Time PCR

Abstract

Citrus huanglongbing (HLB) is caused by the phloem-limited and psyllid-vectored Candidatus Liberibacter spp. and is a destructive disease of citrus that is rapidly increasing in importance. The disease was reported recently in the principle citrus-producing areas of São Paulo, Brazil in 2004 and in Florida in 2005. A variety of laboratory methods have been developed to confirm a symptom-based disease diagnosis or for the detection or identification of the pathogen; however, no quantitative information has been available on the pathogen titer in either host or vector interactions because the pathogen remains unculturable in artificial media. We previously developed a quantitative polymerase chain reaction (PCR)-based assay for detection of Ca. Liberibacter spp. and, in this study, we evaluated the effects of sample composition on quantification of the pathogen in citrus plants by TaqMan real-time PCR. Standard curves were established using cloned plasmids containing target DNA from the pathogen and with total DNA samples from field-grown HLB-infected citrus plants. Regression analysis showed that a standard curve established with DNA extracted from naturally infected field-grown plants was more accurate than the standard curve constructed from plasmids containing the amplification targets as cloned inserts. Nontarget DNA and putative PCR inhibitors from citrus plants decreased the sensitivity and the amplification efficiency of real-time PCR when plasmids provided the template target in “spiked” healthy citrus DNA extracts. This effect varied among plant tissue types, citrus species, and geographic locations. Based on these sample effects, a universal standard curve has been established for quantification of the pathogen in various citrus tissues of different citrus species planted in different geographic locations. Sample storage at 4°C for 2 months prior to PCR assay did not affect subsequent quantification of the pathogen. The validated quantitative real-time PCR method and the universal standard curve will be very useful for studies of host–pathogen interactions and epidemiology, and in the development of control strategies for the disease.

Diversity and Plasticity of the Intracellular Plant Pathogen and Insect Symbiont “Candidatus Liberibacter asiaticus” as Revealed by Hypervariable Prophage Genes with Intragenic Tandem Repeats

“…Liberibacter asiaticus" positive with threshold cycle (C T ) values of between 17 and 29 by 16S rRNA gene-based real-time PCR (Table 2), were equivalent to "Ca. Liberibacter asiaticus" populations ranging between 2.95 ϫ 10 8 and 1.08 ϫ 10 5 bacterial cells/l DNA extracts based on the grand universal regression equation Y ϭ 13.82 Ϫ 0.2866X, where X is the mean C T value and Y is the log concentration of the target DNA copies (32). All the Florida "Ca.…”

In planta multiplication and graft transmission of ‘Candidatus Liberibacter asiaticus’ revealed by Real-Time PCR

“…This factor compensates for the use of 0.25 g in the extraction, and 25% of the extract obtained. Although recently Kim and Wang (2009) determined three sets of ribosomal genes in CLas our data were not corrected against the bacterial concentration obtained for the already published papers (Li et al 2006(Li et al , 2007(Li et al , 2008Manjunath et al 2008;Tatineni et al 2008), which assumed one copy of 16S rRNA gene in the CLas genome.…”

Development and systematic validation of qPCR assays for rapid and reliable differentiation of Xylella fastidiosa strains causing citrus variegated chlorosis

“…Standard linear regressions (Y =a+bX) of the log concentration of the template copies (Y) versus the mean Ct values (X) were obtained. PCR amplification efficiency (AE) was estimated from the slopes of the standard curves using the equation AE = 10 −slope −1 (Li et al, 2008). If necessary, analysis of variance was performed on the means of Ct values and, when significant, this was followed by a means comparison test (Tukey's) using a confidence level of P =0.05.…”