Complete Genome Sequence of Citrus Huanglongbing Bacterium, ‘ Candidatus Liberibacter asiaticus’ Obtained Through Metagenomics

USDA-ARS-USHRL, Fort Pierce, FL 34945, U.S.A.
Molecular Plant-Microbe Interactions (Impact Factor: 3.94). 09/2009; 22(8):1011-20. DOI: 10.1094/MPMI-22-8-1011
Source: PubMed


Citrus huanglongbing is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with a low-titer, phloem-limited infection by any of three uncultured species of alpha-Proteobacteria, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. A complete circular 'Ca. L. asiaticus' genome has been obtained by metagenomics, using the DNA extracted from a single 'Ca. L. asiaticus'-infected psyllid. The 1.23-Mb genome has an average 36.5% GC content. Annotation revealed a high percentage of genes involved in both cell motility (4.5%) and active transport in general (8.0%), which may contribute to its virulence. 'Ca. L. asiaticus' appears to have a limited ability for aerobic respiration and is likely auxotrophic for at least five amino acids. Consistent with its intracellular nature, 'Ca. L. asiaticus' lacks type III and type IV secretion systems as well as typical free-living or plant-colonizing extracellular degradative enzymes. 'Ca. L. asiaticus' appears to have all type I secretion system genes needed for both multidrug efflux and toxin effector secretion. Multi-protein phylogenetic analysis confirmed 'Ca. L. asiaticus' as an early-branching and highly divergent member of the family Rhizobiaceae. This is the first genome sequence of an uncultured alpha-proteobacteria that is both an intracellular plant pathogen and insect symbiont.

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Available from: Allan W Dickerman, Sep 29, 2015
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    • "However, for detection of HLB associated Las, LAMP has not been used widely so far. The availability of the full genome sequence of Las (Duan et al., 2009) has enabled researchers to evaluate other regions of the bacterium that are more suitable for PCR-based detection technologies (Morgan et al., 2012). We have developed a rapid, cost-effective, easy to operate, and field deployable technique to detect Las in psyllids. "
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    ABSTRACT: ‘Candidatus Liberibacter asiaticus’ (Las), associated with citrus huanglongbing (HLB or citrus greening) is spread by Diaphorina citri Kuwayama, the Asian citrus psyllid. Psyllids can be tested to assess the prevalence of Las in a population and for early detection of HLB in new areas being invaded by the psyllid. In some cases, large numbers of psyllids may need to be tested, thus there is a need for rapid and inexpensive field detection methodology. We report here on the development of a field detection kit for testing psyllids for Las using loop-mediated amplification technology (LAMP). Six samples with pools of 1–10 psyllids plus a positive and negative control can be tested at a time in about 30 min; 10 min for crude extraction and 20 min for target DNA amplification. The LAMP assays are conducted in a Smart-DART™ detection unit which is operated from an Android device. The LAMP detection method for Las is about 100 times more sensitive than the traditional real time PCR method. In addition to field testing of psyllids for Las, the methodology was validated as effective for identifying Las in plant DNA extractions. In California, where the psyllid has only recently invaded, participation of a large number of growers and extension workers in field detection may facilitate rapid containment efforts should Las be found. In areas where the disease epidemic is already in the initial stages, large scale testing can be helpful for effective disease management. The LAMP technology lends itself well in such situations.
    Crop Protection 11/2014; 68. DOI:10.1016/j.cropro.2014.10.026 · 1.49 Impact Factor
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    • "The presence of tricarboxylic acid (TCA) cycle genes in CLas genome indicated that CLas can use a wide range of amino acids to produce energy [32]. CLas can metabolize glutamate, alanine, aspartate, glycine, serine, threonine, methionine, cysteine, arginine, proline, histidine, tyrosine, phenylalanine, and tryptophan [32]. All of the previous amino acids were detected in the phloem sap. "
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    ABSTRACT: Through utilizing the nutrient-rich phloem sap, sap feeding insects such as psyllids, leafhoppers, and aphids can transmit many phloem-restricted pathogens. On the other hand, multiplication of phloem-limited, uncultivated bacteria such as Candidatus Liberibacter asiaticus (CLas) inside the phloem of citrus indicates that the sap contains all the essential nutrients needed for the pathogen growth. The phloem sap composition of many plants has been studied; however, to our knowledge, there is no available data about citrus phloem sap. In this study, we identified and quantified the chemical components of phloem sap from pineapple sweet orange. Two approaches (EDTA enhanced exudation and centrifugation) were used to collect phloem sap. The collected sap was derivatized with methyl chloroformate (MCF), N-methyl-N- [tert-butyl dimethylsilyl]-trifluroacetamide (MTBSTFA), or trimethylsilyl (TMS) and analyzed with GC-MS revealing 20 amino acids and 8 sugars. Proline, the most abundant amino acid, composed more than 60% of the total amino acids. Tryptophan, tyrosine, leucine, isoleucine, and valine, which are considered essential for phloem sap-sucking insects, were also detected. Sucrose, glucose, fructose, and inositol were the most predominant sugars. In addition, seven organic acids including succinic, fumaric, malic, maleic, threonic, citric, and quinic were detected. All compounds detected in the EDTA-enhanced exudate were also detected in the pure phloem sap using centrifugation. The centrifugation technique allowed estimating the concentration of metabolites. This information expands our knowledge about the nutrition requirement for citrus phloem-limited bacterial pathogen and their vectors, and can help define suitable artificial media to culture them.
    PLoS ONE 07/2014; 9(7):e101830. DOI:10.1371/journal.pone.0101830 · 3.23 Impact Factor
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    • "The combination of this isothermal DNA amplification technique with LFD devices has proven to be robust and successful in field-capable molecular diagnostics [20-22]. The recent sequencing of Las genome has uncovered new DNA sequences that can be used for pathogen detection through DNA amplification technologies [23]. Using an “in silico” approach, we found a hypothetical protein coding sequence, CLIBASIA_05175 [GenBank: ACT57606.1], "
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    ABSTRACT: Citrus Huanglongbing (HLB) is the most devastating bacterial citrus disease worldwide. Three Candidatus Liberibacter species are associated with different forms of the disease: Candidatus Liberibacter asiaticus, Candidatus Liberibacter americanus and Candidatus Liberibacter africanus. Amongst them, Candidatus Liberibacter asiaticus is the most widespread and economically important. These Gram-negative bacterial plant pathogens are phloem-limited and vectored by citrus psyllids. The current management strategy of HLB is based on early and accurate detection of Candidatus Liberibacter asiaticus in both citrus plants and vector insects. Nowadays, real time PCR is the method of choice for this task, mainly because of its sensitivity and reliability. However, this methodology has several drawbacks, namely high equipment costs, the need for highly trained personnel, the time required to conduct the whole process, and the difficulty in carrying out the detection reactions in field conditions. A recent DNA amplification technique known as Loop Mediated Isothermal Amplification (LAMP) was adapted for the detection of Candidatus Liberibacter asiaticus. This methodology was combined with a Lateral Flow Dipstick (LFD) device for visual detection of the resulting amplicons, eliminating the need for gel electrophoresis. The assay was highly specific for the targeted bacterium. No cross-reaction was observed with DNA from any of the other phytopathogenic bacteria or fungi assayed. By serially diluting purified DNA from an infected plant, the sensitivity of the assay was found to be 10 picograms. This sensitivity level was proven to be similar to the values obtained running a real time PCR in parallel. This methodology was able to detect Candidatus Liberibacter asiaticus from different kinds of samples including infected citrus plants and psyllids. Our results indicate that the methodology here reported constitutes a step forward in the development of new tools for the management, control and eradication of this destructive citrus disease. This system constitutes a potentially field-capable approach for the detection of the most relevant HLB-associated bacteria in plant material and psyllid vectors.
    BMC Microbiology 04/2014; 14(1):86. DOI:10.1186/1471-2180-14-86 · 2.73 Impact Factor
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