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PubMed1997

Production and evaluation of non-radioactive probes for the detection of the two 'Candidatus Liberobacter' species associated with citrus huanglongbing (greening).

The production and evaluation of non-radioactive probes for the detection of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter africanum', the two bacterial species associated with citrus huanglongbing (greening) disease is described. Two DNA fagments, In 2.6 and AS 1.7, obtained previously from the beta operons of 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter africanum', respectively, were the starting materials for production of the two non-radioactive probes. These digoxigenin (DIG)-labelled probes were generated by PCR incorporation of DIG-11-dUTP, yielding In 1.7-DIG and AS 1.7-DIG. Probe In 1.7-DIG was hybridized with DNAs extracted from 24 field-collected samples in Bali (Indonesia). The membrane on which the DNAs were blotted was first hybridized with radioactive probe 32P-In 2.6. After the hybridization results were recorded, the radioactive probe was removed, and the membrane hybridized with DIG-labelled probe In 1.7-DIG. Identical results were obtained for 23 samples. One sample was positive with the DIG-labelled probe and negative with the 32P-labelled probe. However, cross-hybridization of In 1.7-DIG with DNA from L. africanum was higher than that obtained with the radioactive probe. This cross-hybridization could be eliminated by raising the temperature of the stringent washing step. No field samples from Africa being available, probe AS 1.7-DIG was dot-blot hybridized against DNAs extracted from leaves of greenhouse-kept citrus plants from different geographical origins and infected with one or other Liberobacter species. The data showed that AS 1.7-DIG hybridized with L. africanum with a sensitivity equivalent to that of the radioactive probe.

Citrus↗
PubMed1999

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

Huanglongbing, previously called greening, is a destructive disease of citrus in Asia and Africa. It is caused by an uncultured, phloem-restricted bacterium for which two species Candidatus Liberobacter asiaticum and Candidatus Liberobacter africanum have been characterized. In 1996, a polymerase chain reaction (PCR) detection method based on the amplification of the 16S ribosomal operon was developed and proved to be efficient for the detection of both liberobacter species. However, in order to distinguish between Candidatus Liberobacter asiaticum and Candidatus Liberobacter africanum, digestion of the amplified DNA with XbaI was required. We have now developed a new PCR detection method based on the amplification of ribosomal protein genes which allows direct identification of the liberobacter species by the size of the amplified DNA. This PCR method is as specific, and at least as sensitive as the previously described one for detection of the two liberobacter species

Animals↗
PubMed1999

Isolation of DNA from the uncultured "Candidatus Liberobacter" species associated with citrus Huanglongbing by RAPD.

"Candidatus Liberobacter," the uncultured bacterium associated with citrus Huanglongbing (HLB) disease, is an alpha-Proteobacteria, and two species, "Candidatus L. africanum" and "Candidatus L. asiaticum, " have been characterized by sequence analysis of the 16S rDNA and beta operon (rplKAJL-rpoBC) genes. These genes were isolated by PCR and random cloning of DNA from infected plants. However, this strategy is laborious and allowed selection of only three Liberobacter DNA fragments. In this paper, we described isolation of additional genes using Random Amplified Polymorphic DNA (RAPD). In total, 102 random 10-mer primers were used in PCR reactions on healthy and Liberobacter-infected plant DNA. Eight DNA bands amplified from infected plant DNA were cloned and analyzed. Six of them were found to be part of the Liberobacter genome by sequence and hybridization experiments. On these DNA fragments, four genes were identified: nusG, pgm, omp, and a hypothetical protein gene. These results indicate that RAPD can be used to clone DNA of uncultured organisms.

Amino Acid Sequence↗
PubMed2005

Citrus huanglongbing in São Paulo State, Brazil: PCR detection of the 'Candidatus' Liberibacter species associated with the disease.

Symptoms of huanglongbing (HLB), one of the most serious diseases of citrus in Asia and Africa, have been noticed in March 2004 in the Araraquara region of São Paulo State, Brazil. HLB has not been reported previously from America. The causal HLB bacteria, Candidatus Liberibacter africanus in Africa and Candidatus Liberibacter asiaticus in Asia, can be detected in symptomatic citrus leaves by PCR amplification of their 16S rDNA with previously described primers. When this technique was applied to 43 symptomatic leaf samples from the Araraquara region, all PCR reactions were negative. This suggested that a new pathogen, not detected by the above primers, could be involved in HLB in the State of São Paulo. Indeed, by using universal primers for amplification of bacterial 16S rDNA, a new liberibacter species, Candidatus Liberibacter americanus, has recently been identified. Specific primers for PCR amplification of the 16S rDNA of Ca. L. americanus have been selected. Using these primers, the new liberibacter could be detected in 214 symptomatic leaf samples tested. The leaves of two additional samples were infected with Candidatus Liberibacter asiaticus, and two further samples contained both Ca. L. americanus and Ca. L. asiaticus. The samples came from 47 farms in 35 municipalities. The psyllid vector of Ca. L. asiaticus, Diaphorina citri, is established in South, Central, and North America (Florida and Texas). Ca. L. americanus could be detected by PCR in several batches of D. citri psyllids collected on symptomatic sweet orange trees infected with Ca. L. americanus, strongly suggesting that D. citri is the vector of Ca. L. americanus. The results reported here confirm the presence of HLB in the State of São Paulo. Ca. L. americanus is the most widely distributed pathogen.

Base Sequence↗
PubMed2006

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

Citrus huanglongbing (HLB, ex greening) is one of the most serious diseases of citrus. Different forms of the disease are caused by different Candidatus Liberobacter species, Candidatus Liberibacter asiaticus (Las), Ca. L. africanus (Laf) and Ca. L. americanus (Lam). The pathogen is transmitted by psyllid insects and by budding with contaminated plant materials. The vector psyllid Diaphorina citri can transmit both Las and Lam. Establishment of this vector into Florida, reports of Lam and Las in Brazil in 2004, and recent confirmation of HLB in Florida in September 2005 is of great concern to the citrus industry. Research on HLB has been hampered by the unculturable nature of the causal bacterium in artificial media. It has also been difficult to detect and identify the pathogens, possibly because of low concentration and uneven distribution in host plants and vector psyllids. In this study, we developed quantitative TaqMan PCR using 16S rDNA-based TaqMan primer-probe sets specific to the different Ca. Liberobacter spp. An additional primer-probe set based on plant cytochrome oxidase (COX) was used as a positive internal control to assess the quality of the DNA extracts. The assays do not cross-react with other pathogens or endophytes commonly resident in citrus plants, and are very sensitive. HLB pathogen DNA was successfully amplified from the equivalent of 20 ng of midrib tissue from symptomatic leaves. The consistent results of the assays with DNA extracted from plants infected by various Ca. Liberibacter species grown in greenhouses and in the field demonstrated a degree of reproducibility for these TaqMan assays. Inhibitors of the PCR that are frequently present in plant extracts did not affect the assay results. The population of the pathogens was estimated to be 5 x 10(7) and 2 x 10(6) cells/g of fresh midribs of symptomatic sweet orange leaves infected by Las and Lam, respectively. The ratio of pathogen DNA to host plant DNA was estimated by to be 1:13,000 (w/w) and 1:1000 (c/c: target copy/target copy) in DNA extracts obtained by a standard CTAB method. Our rapid, sensitive and specific TaqMan PCR assay for the detection, identification and quantification of Ca. Liberibacter species has been successfully used in the confirmation of HLB caused by Las in Florida, and will be very useful for a broad range of research programs as well as the regulatory response and management of HLB disease.

Alphaproteobacteria↗
PubMed2005

'Candidatus Liberibacter americanus', associated with citrus huanglongbing (greening disease) in São Paulo State, Brazil.

Symptoms of huanglongbing (HLB) were reported in São Paulo State (SPS), Brazil, in March 2004. In Asia, HLB is caused by 'Candidatus Liberibacter asiaticus' and in Africa by 'Candidatus Liberibacter africanus'. Detection of the liberibacters is based on PCR amplification of their 16S rRNA gene with specific primers. Leaves with blotchy mottle symptoms characteristic of HLB were sampled in several farms of SPS and tested for the presence of liberibacters. 'Ca. L. asiaticus' was detected in a small number of samples but most samples gave negative PCR results. Therefore, a new HLB pathogen was suspected. Evidence for an SPS-HLB bacterium in symptomatic leaves was obtained by PCR amplification with universal primers for prokaryotic 16S rRNA gene sequences. The amplified 16S rRNA gene was cloned and sequenced. Sequence analysis and phylogeny studies showed that the 16S rRNA gene possessed the oligonucleotide signatures and the secondary loop structure characteristic of the alpha-Proteobacteria, including the liberibacters. The 16S rRNA gene sequence phylogenetic tree showed that the SPS-HLB bacterium clustered within the alpha-Proteobacteria, the liberibacters being its closest relatives. For these reasons, the SPS-HLB bacterium is considered a member of the genus 'Ca. Liberibacter'. However, while the 16S rRNA gene sequences of 'Ca. L. asiaticus' and 'Ca. L. africanus' had 98.4% similarity, the 16S rRNA gene sequence of the SPS-HLB liberibacter had only 96.0% similarity with the 16S rRNA gene sequences of 'Ca. L. asiaticus' or 'Ca. L. africanus'. This lower similarity was reflected in the phylogenetic tree, where the SPS-HLB liberibacter did not cluster within the 'Ca. L asiaticus'/'Ca. L. africanus group', but as a separate branch. Within the genus 'Candidatus Liberibacter' and for a given species, the 16S/23S intergenic region does not vary greatly. The intergenic regions of three strains of 'Ca. L. asiaticus', from India, the People's Republic of China and Japan, were found to have identical or almost identical sequences. In contrast, the intergenic regions of the SPS-HLB liberibacter, 'Ca. L. asiaticus' and 'Ca. L. africanus' had quite different sequences, with similarity between 66.0 and 79.5%. These results confirm that the SPS-HLB liberibacter is a novel species for which the name 'Candidatus Liberibacter americanus' is proposed. Like the African and the Asian liberibacters, the 'American' liberibacter is restricted to the sieve tubes of the citrus host. The liberibacter could also be detected by PCR amplification of the 16S rRNA gene in Diaphorina citri, the psyllid vector of 'Ca. L. asiaticus', suggesting that this psyllid is also a vector of 'Ca. L. americanus' in SPS. 'Ca. L. americanus' was detected in 216 of 218 symptomatic leaf samples from 47 farms in 35 municipalities, while 'Ca. L. asiaticus' was detected in only 4 of the 218 samples, indicating that 'Ca. L. americanus' is the major cause of HLB in SPS.

Brazil↗
PubMed1997

Comparison of the 16S/23S ribosomal intergenic regions of "Candidatus Liberobacter asiaticum" and "Candidatus Liberobacter africanum," the two species associated with citrus huanglongbing (greening) disease.

16S/23S intergenic spacer regions from the rRNA operons of two strains of "Candidatus Liberobacter asiaticum" and one strain of "Candidatus Liberobacter africanum" were cloned and sequenced. The intergenic spacers of the two "Candidatus L. asiaticum" strains studied are identical and contain the genes for isoleucine tRNA (tRNA(Ile)) and alanine tRNA (tRNA(Ala)) separated by 11 nucleotides. The intergenic spacer of the "Candidatus L. africanum" strain contains only one tRNA gene (tRNA(Ala)). The level of homology between the intergenic spacers of the two liberobacter species is 79.46%. Ribosomal operons with 16S/23S spacer regions other than those studied might be present in the two "Candidatus Liberobacter" species.

Base Sequence↗
PubMed2026

High-efficiency genome-editing, transgene evaluation, and antimicrobial efficacy testing using Citrus medica L. hairy roots.

Huanglongbing (HLB) disease, associated with the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), has a significant impact on citrus production worldwide. Conventional biochemical and genetic evaluation studies to identify potential disease resistance strategies have been mainly hindered due to the inability to culture CLas in a defined medium and the general recalcitrance of Citrus cultivars (grapefruits and oranges) to Agrobacterium-mediated plant transformation. We previously demonstrated the utility of plant hairy roots to co-cultivate CLas. In this study, we developed a hairy root transformation system using citron (Citrus medica L.), which is highly amenable to Rhizobium-mediated hairy root transformation. The explant survival and hairy root transformation efficiencies were up to 100% and 73%, respectively, and transgenic roots can be attained in as little as 30-60 days. We demonstrate the utility of this citron-based hairy root transformation for rapid CRISPR/Cas9-mediated gene editing, transgene evaluation, and antimicrobial efficacy testing. The citron-based hairy root transformation system will significantly help the research community to speed-track the assessment of potential HLB disease resistance strategies.

Citrus↗
PubMed2026

Dual-gRNA CRISPR/Cas9 Deletion of CsDMR6 in Sweet Orange Supported by Improved In Vitro Regeneration.

Huanglongbing (HLB), caused by Candidatus Liberibacter spp., remains the most destructive disease affecting citrus worldwide. To support host-directed genome-editing strategies aimed at reducing susceptibility, we optimized key regeneration steps in Citrus sinensis and validated a dual-gRNA CRISPR/Cas9 approach targeting the susceptibility gene CsDMR6. Juvenile explants of 'Valencia' and hybrid genotypes (CsH1-CsH3) were successfully established in vitro, and shoot elongation was markedly improved by supplementing Citrus Shoot Multiplication (CiSM) medium with 1 mg L-1 GA3. Callus induction was most efficient in Citrus Callus Induction (CiCM) medium under dark conditions, while a 48 h NAA pulse (100 µM) significantly enhanced rooting, increasing efficiencies to 37.1% in 'Valencia' and 52.9% in CsH1. Two guide RNAs targeting conserved regions of CsDMR6 were designed and shown to be identical across all evaluated genotypes. The dual-gRNA cassette was assembled into a CRISPR/Cas9 geminivirus-based vector and transiently delivered into sweet orange leaf tissue via Agrobacterium. GFP fluorescence verified construct expression, and PCR amplification across the target region produced a diagnostic ~447 bp fragment corresponding to the expected ~5.8 kb deletion. Sanger sequencing confirmed precise junction formation between the two cut sites. These results demonstrate efficient large-fragment deletion of CsDMR6 in sweet orange and establish an experimentally validated, genotype-compatible regeneration and editing platform. This study provides a transient validation of the dual-gRNA system and establishes the technical foundation required for future stable, non-transgenic edited lines. Together, these advances support the downstream functional evaluation of CsDMR6 loss-of-function alleles under HLB pressure.

CRISPR/Cas9↗
PubMed2000

Genomic characterization of a liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape Province of South Africa. Proposal of 'Candidatus Liberibacter africanus subsp. capensis'.

In 1994, the uncultured phloem-restricted bacteria of citrus huanglongbing (ex-greening) disease in Asia and Africa were characterized as 'Candidatus Liberobacter asiaticum' and 'Candidatus Liberobacter africanum', respectively. Following the rules of the International Code of Nomenclature of Bacteria, the two bacterial species have now been renamed 'Candidatus Liberibacter asiaticus' and 'Candidatus Liberibacter africanus'. A third liberibacter was detected by PCR in an ornamental rutaceous tree, Cape chestnut (Calodendrum capense), in South Africa. The new liberibacter was characterized by serology and from the sequences of its 16S rDNA, intergenic 16S/23S rDNA and ribosomal protein genes of the beta operon. Phylogenetic analysis showed that the liberibacter present in C. capense differed from the two previously described liberibacter species from citrus and that it was more closely related to 'Candidatus Liberibacter africanus' than to 'Candidatus Liberibacter asiaticus'. It is proposed that the liberibacter from C capense be assigned a subspecies status, 'Candidatus Liberibacter africanus subsp. capensis'.

Alphaproteobacteria↗
PubMed2005

Diversity of "Candidatus Liberibacter asiaticus," based on the omp gene sequence.

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.

Asia↗