Isolation of DNA from the Uncultured “Candidatus Liberobacter” Species Associated with Citrus Huanglongbing by RAPD

Hocquellet, Agnès; Bové, Joseph M.; Garnier, M.

doi:10.1007/pl00006783

Cited by 16 publications

(11 citation statements)

References 27 publications

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“…Detection and identification of the two "Ca. Liberibacter" species were achieved by 16S rRNA gene-based PCR-RFLP analysis (20), as well as PCR amplification of ribosomal protein genes (18). While these tools allowed specific detection of the two HLB "Ca.…”

Section: Discussionmentioning

confidence: 99%

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Diversity of “ Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Bastianel¹,

Garnier-Semancik²,

Renaudin³

et al. 2005

Appl Environ Microbiol

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Huanglongbing (yellow dragon disease) is a destructive disease of citrus. The etiological agent is a noncultured, phloem-restricted alpha-proteobacterium, "Candidatus Liberibacter africanus" in Africa and "Candidatus Liberibacter asiaticus" in Asia. In this study, we used an omp-based PCR-restriction fragment length polymorphism (RFLP) approach to analyze the genetic variability of "Ca. Liberibacter asiaticus" isolates. By using five different enzymes, each the 10 isolates tested could be associated with a specific combination of restriction profiles. The results indicate that the species "Ca. Liberibacter asiaticus," even within a given region, may comprise several different variants. Thus, omp-based PCR-RFLP analysis is a simple method for detecting and differentiating "Ca. Liberibacter asiaticus" isolates.

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Section: Discussionmentioning

confidence: 99%

“…To further characterize the liberibacters at the molecular level, additional DNA fragments have been isolated by the randomly amplified polymorphic DNA method (18). Sequence analyses of six such fragments showed that they were part of the "Ca.…”

mentioning

confidence: 99%

Diversity of “ Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Bastianel¹,

Garnier-Semancik²,

Renaudin³

et al. 2005

Appl Environ Microbiol

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“…In-2.6 and AS-1.7 have been used as specific hybridization probes for the detection of the Asian and African liberibacter strains, respectively (Villechanoux et al, 1992;Hocquellet et al, 1997). They have served to design PCR primers rplA2 and rplJ5 for detection of liberibacters by amplification of ribosomal protein genes (Hocquellet et al, 1999). Finally, comparison of the sequences of In-2.6 and AS-1.7 confirmed that the Asian and African liberibacters represent two distinct species (Planet et al, 1995).…”

Section: Introductionmentioning

confidence: 92%

The tufB-secE-nusG-rplKAJL-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation

Teixeira¹,

Eveillard²,

Sirand‐Pugnet³

et al. 2008

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLO

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The rplKAJL-rpoBC operon or b operon is a classic bacterial gene cluster, which codes for proteins K, A, J and L of the large ribosomal subunit, as well as proteins B (b subunit) and C (b9 subunit) of RNA polymerase. In the early 1990s, the operon was obtained as a 2.6 kbp DNA fragment (In-2.6) by random cloning of DNA from periwinkle plants infected with the Poona (India) strain of the huanglongbing agent, later named 'Candidatus (Ca.) Liberibacter asiaticus'. DNA from periwinkle plants infected with the Nelspruit strain (South Africa) of 'Ca. L. africanus' was amplified with a primer pair designed from In-2.6 and yielded, after cloning and sequencing, a 1.7 kbp DNA fragment (AS-1.7) of the b operon of 'Ca. L. africanus'. The b operon of the American liberibacter, as well as the three upstream genes (tufB, secE, nusG), have now also been obtained by the technique of chromosome walking and extend over 4673 bp, comprising the following genes: tufB, secE, nusG, rplK, rplA, rplJ, rplL and rpoB. The sequence of the b operon was also determined for a Brazilian strain of 'Ca. L. asiaticus', from nusG to rpoB (3025 bp), and was found to share 99 % identity with the corresponding b operon sequences of an Indian and a Japanese strain. Finally, the b operon sequence of 'Ca. L. africanus' was extended from 1673 bp (rplA to rpoB) to 3013 bp (nusG to rpoB), making it possible to compare the b operon sequences of the African, Asian and American liberibacters over a length of~3000 bp, from nusG to rpoB. While 'Ca. L. africanus' and 'Ca. L. asiaticus' shared 81.2 % sequence identity, the percentage for 'Ca. L. americanus' and 'Ca. L. africanus' was only 72.2 %, and identity for 'Ca. L. americanus' and 'Ca. L. asiaticus' was only 71.4 %. The~3000 bp nusG-rpoB sequence was also used to construct a phylogenetic tree, and this tree was found to be identical to the known 16S rRNA gene sequence-based tree. These results confirm earlier findings that 'Ca. L. americanus' is a distinct liberibacter, more distantly related to 'Ca. L. africanus' and 'Ca. L. asiaticus' than 'Ca. L. africanus' is to 'Ca. L. asiaticus'. The dates of speciation have also been estimated.

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“…Other molecular studies on the proteobacteria have involved PCR detection based on the 16S ribosomal operon followed by digestion of the PCR products to distinguish between the African and Asiatic strain. Later an improvement on this protocol involved PCR-based detection of the ribosomal protein genes of the β operon without concomitant enzymatic digestion (Hocquellet et al 1999a;Hocquellet et al 1999b;Lan et al 2004;Okuda et al 2005;Li et al 2007;Li et al 2008;Tatineni et al 2008). Bastianel et al (2005) described an omp-based PCR-RFLP analysis method for detecting and differentiating 'Candidatus Liberibacter asiaticus' isolates.…”

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confidence: 99%

Molecular characterization of ‘Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

Magomere¹,

Obukosia²,

Mutitu³

et al. 2009

Electron. J. Biotechnol.

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Isolation of DNA from the Uncultured “Candidatus Liberobacter” Species Associated with Citrus Huanglongbing by RAPD

Cited by 16 publications

References 27 publications

Diversity of “ Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

Diversity of “ Candidatus Liberibacter asiaticus,” Based on the omp Gene Sequence

The tufB-secE-nusG-rplKAJL-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation

Molecular characterization of ‘Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

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