Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing

Li, Wenbin; Hartung, John; Lévy, L.

doi:10.1016/j.mimet.2005.10.018

Cited by 734 publications

(682 citation statements)

References 28 publications

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“…The mechanism behind the phloem disorder is still unknown. It has been speculated that the HLB pathogen is present in the citrus phloem at low titer (Li et al 2006). However, with the exception of some microscopic studies, not enough is understood regarding the precise bacterial concentration in planta (Bové 2006).…”

Section: Introductionmentioning

confidence: 99%

“…DNA hybridisation methods are time-consuming and labour-intensive. Recently, quantitative real-time PCR based on the primers from 16S rDNA (Li et al 2006;Teixeira et al 2008) and rplKAJL-rpoBC (β-operon) (Hocquellet et al 1997;) have been used for detection and quantification of the HLB pathogen. However, these assays could not differentiate between viable and dead cells.…”

Section: Introductionmentioning

confidence: 99%

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Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

et al. 2009

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Citrus Huanglongbing (HLB) is a devastating disease of citrus known to be associated with a fastidious, phloem-limited Gram-negative, yet to be cultured bacterium in the genus Candidatus Liberibacter. In the present study we have developed a method to quantify viable Candidatus Liberibacter asiaticus (Las) with the aid of ethidium monoazide (EMA) which can differentiate live from dead cells. First, calibration curves were developed with the aid of quantitative real-time PCR (QPCR) by using a plasmid template consisting of a 703 bp DNA fragment of rplKAJL-rpoBC (β-operon) region. Standard equations were then developed to quantify Las genome equivalents in citrus, periwinkle, and Asian citrus psyllid, Diaphorina citri. To overcome the limitation of quantitative PCR in discriminating between live and dead bacterial cells, EMA was used to inhibit the amplification of DNA from the dead cells of Las in plant samples. By using the standard equations and EMA-QPCR methods developed in this study, we found that the proportion of viable cells in citrus and periwinkle ranged from 17-31% and 16-28%, respectively. It was determined that a minimum bacterial concentration is required for HLB symptom development by quantifying the population of Las in symptomatic and asymptomatic leaves. The EMA-QPCR methodology developed in the present study should provide an accurate assessment of viable HLB pathogen, providing a tool to investigate disease epidemiology and thus act as a crucial component for disease assessment and management.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

et al. 2009

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“…DNA extracts from C. capense specimens were tested for Liberibacters and for LafC specifically using a the labeled probe (HLBp) and reverse primer (HLBr) from the real-time PCR of Li et al (2006) but as the forward primer LibUF, to a conserved region of known Liberibacters (5'-GGCAGGCCTAACACATGC-3') ("Generic Liberibacter real-time PCR") or a LafC specific primer, LafC F (5'-ATTGCGCGTATCGAATACGACG-3') and using as template 1µl of the DNA extract. The specificity of the LafC specific and generic Liberibacter PCR were tested against total DNA extracts containing Laf, LafC, "Ca.…”

Section: Liberibacter Real-time Pcr Amplificationmentioning

confidence: 99%

“…Real-time PCR was performed using a Lightcycler® 1.5 (Roche, Mannheim, Germany) capillary-based thermocycler. Lightcycler® Taqman® Master kits were used along with the LibUF, LafC primers and probes and conditions as described (by Li et al 2006). A positive/negative crossing threshold (Ct) of 30 was used after parallel tests showed that samples with Ct values of 30 in either the LafC-specific and Generic Liberibacter systems no longer yielded amplicons in conventional PCR.…”

Section: Liberibacter Real-time Pcr Amplificationmentioning

confidence: 99%

Widespread occurrence of “Candidatus liberibacter africanus subspecies capensis” in Calodendrum capense in South Africa

2012

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Recent studies in citrus orchards confirmed that Citrus Greening, a heat sensitive citrus disease, similar to Huanglongbing (HLB), is associated with the presence of "Candidatus Liberibacter africanus" (Laf) in South Africa. Neither "Candidatus Liberibacter asiaticus" (Las), associated with HLB; "Candidatus Liberibacter americanus"; nor "Candidatus Liberibacter africanus ssp. capense specimens, 43 were infected with LafC, but none of the citrus trees were infected with LafC. Based on the results of this it appears that natural spread of LafC to citrus does not occur.

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“…The purified DNA amplification product from one adult specimen collected in Kamichu was directly sequenced in both directions at the Australian Genome Research Facility Ltd with automated sequencing using an Applied Biosystems 3730xl capillary sequencer (www.agrf.org.au) to confirm the identity of the amplified fragment. Real-time PCR was performed as per Li et al (2006) with an internal control for monitoring the quality of psyllid DNA extraction coding for the wingless gene using the primers and probe (DCF, DCR and DCP) described by Manjunath et al (2008).…”

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confidence: 99%

First report of ‘Candidatus Liberibacter asiaticus’ in Diaphorina communis

Donovan

Beattie

Chambers

et al. 2011

Australasian Plant Dis. Notes

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Huanglongbing (HLB) or citrus greening is one of the most destructive diseases of citrus in the world and one of the major factors limiting citrus production in south east Asia including Bhutan. The presence of 'Candidatus Liberibacter asiaticus', associated with the Asiatic form of HLB, was confirmed by conventional and real-time PCR in adults of the black psyllid, Diaphorina communis Mathur. This is the first formal detection of 'Ca. L. asiaticus' in D. communis, and the first detection of the pathogen in a psyllid other than D. citri Kuwayama in Asia, excluding Arabia. This study is also the first to report the presence of D. communis in Bhutan.

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Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing

Cited by 734 publications

References 28 publications

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Quantification of viable Candidatus Liberibacter asiaticus in hosts using quantitative PCR with the aid of ethidium monoazide (EMA)

Widespread occurrence of “Candidatus liberibacter africanus subspecies capensis” in Calodendrum capense in South Africa

First report of ‘Candidatus Liberibacter asiaticus’ in Diaphorina communis

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