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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    • "Research on Las genomics has provided researchers with complete sequence data to develop more sensitive molecular markers to study the population biology of the HLB pathogens (Duan et al. 2009; Tyler et al. 2009). Chen et al. (2010) were the first to take advantage of this resource and identified a locus containing a single variable tandem repeat; i.e. a microsatellite locus. "
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    ABSTRACT: Citrus huanglongbing (HLB) has become a major disease and limiting factor of production in the citrus areas that have become infected. The destruction to the affected citrus industries has resulted in a tremendous increase to support research that in return has resulted in significant information on both applied and basic knowledge concerning this important disease to the global citrus industry. Recent research indicates the relationship between citrus and the causal agent of HLB is shaped by multiple elements, in which host defense responses may also play an important role. This review is intended to provide an overview of the importance of HLB to a wider audience of plant biologists. Recent advances on host pathogen interactions, population genetics, and vectoring of the causal agent are discussed.
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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.
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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.
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