Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of theβ operon

Hocquellet, Agnès; Toorawa, P.; Bové, J.M.; Garnier, M.

doi:10.1006/mcpr.1999.0263

Cited by 176 publications

(137 citation statements)

References 12 publications

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“…The HLB-specific plasmid template used in this study, pLBA2, was developed by cloning a portion of DNA sequence of the Las rplKAJL-rpoBC (β-operon) region (Hocquellet et al 1999). The resulting 703 bp fragment was amplified as described above using Liberibacter-specific primers A2 and J5 (Hocquellet et al 1999) and DNA was extracted from infected leaf midribs as a template.…”

Section: Cloning and Sequencingmentioning

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: Cloning and Sequencingmentioning

confidence: 99%

“…The resulting 703 bp fragment was amplified as described above using Liberibacter-specific primers A2 and J5 (Hocquellet et al 1999) and DNA was extracted from infected leaf midribs as a template. The gene sequence for rplKAJL-rpoBC (β-operon) was obtained from GenBank (Accession no: AY342001.1).…”

Section: Cloning and Sequencingmentioning

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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“…Ten leaf samples were collected from five trees with and five trees without symptoms, and DNA was isolated from leaf midribs using the method described by Dellaporta et al (1983). DNA was used as template in a simple PCR reaction with specific primers A2/J5 (Hocquellet et al , 1999) that amplify the ribosomal protein gene in the rplKAJL-rpoBC operon of both ' Ca . L. africanum' and ' Ca .…”

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confidence: 99%

First report of a 16SrXIV, ‘Candidatus Phytoplasma cynodontis’ group phytoplasma associated with coconut yellow decline in Malaysia

et al. 2009

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“…DNA extracts were tested using conventional PCR reactions using primers A2 and J5, which were designed to amplify part of the β operon containing the ribosomal protein genes rplA and rplJ of 'Ca. Liberibacter asiaticus' and 'africanus' (Hocquellet et al 1999) and were multiplexed with primers, fD1 and rP2, designed to amplify the 16S rDNA gene of various bacterial species to serve as an amplification (internal) control (Weisberg et al 1991). Negative 'water' controls and known positive samples from leaves and psyllids (collected in Papua New Guinea) were also included.…”

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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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Detection and identification of the two Candidatus Liberobacter species associated with citrus huanglongbing by PCR amplification of ribosomal protein genes of theβ operon

Cited by 176 publications

References 12 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)

First report of a 16SrXIV, ‘Candidatus Phytoplasma cynodontis’ group phytoplasma associated with coconut yellow decline in Malaysia

First report of ‘Candidatus Liberibacter asiaticus’ in Diaphorina communis

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