Complete genome sequence of citrus huanglongbing bacterium, “Candidatus Liberibacter asiaticus” obtaine through metagenomics

USDA-ARS-USHRL, Fort Pierce, FL 34945, U.S.A.
Molecular Plant-Microbe Interactions (Impact Factor: 4.46). 09/2009; 22(8):1011-20. DOI: 10.1094/MPMI-22-8-1011
Source: PubMed

ABSTRACT 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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    • "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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    • "Ct values not determined after 40 cycles were assigned a value of 41. Copy numbers were divided by three to adjust for the three 16s rDNA gene copies present in the Las genome (Duan et al., 2009) and data were expressed as numbers of Las genomes per gram of plant tissue. "
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    ABSTRACT: Candidatus Liberibacter asiaticus (Las) is a phloem-limited bacterium associ-ated with huanglongbing (HLB), one of the most destructive diseases of citrus in Florida and other citrus-producing countries. Natural transmission of Las occurs by the psyllid vector Diaphorina citri, but transmission can also occur through grafting with diseased budwood. As a result of the difficulty of maintaining Las in culture, screening of citrus germplasm for HLB resistance often relies on graft inoculation as the mode of pathogen transmission. This study evaluates transmission efficiencies and HLB progression in graft-inoculated and psyllid-inoculated citrus under greenhouse and natural conditions in the field. Frequencies of transmission in graft-inoculated greenhouse-grown plants varied between experiments and were as high as 90% in susceptible sweet orange plants 6 to 12 months after inoculation. Transmission frequency in a tolerant Citrus 3 Poncirus genotype (US-802) was 31% to 75%. In contrast, transmission of Las after controlled psyllid inoculation did not exceed 38% in any of four experiments in this study. Whereas the time from inoculation to detection of Las by polymerase chain reaction (PCR) was faster in psyllid-inoculated US-802 plants compared with graft-inoculated US-802 plants, it was similar in graft-and psyllid-inoculated sweet orange plants. HLB symptom expression was indistinguishable in graft-and psyllid-inoculated plants but was not always associated with the number of bacteria in affected leaves. The highest number of Las genomes per gram leaf tissue measured in sweet orange plants was one to four 3 10 7 in graft-inoculated plants and one to two 3 10 7 in psyllid-inoculated plants. Highest numbers measured in tolerant US-802 plants were one to three 3 10 6 and two to six 3 10 6 , respectively. Compared with artificial inoculation in a greenhouse setting, natural inoculation of field-grown sweet orange trees occurred at a much slower pace, requiring more than 1 year for infection incidence to reach 50% and a minimum of 3 years to reach 100%. Candidatus Las, a phloem-limited Gram-negative bacterium (Garnier et al., 1984) is the organism associated with citrus HLB in Florida and most other citrus-producing countries around the world (Bové, 2006; Gottwald, 2010). HLB, which is also known as citrus greening or yellow shoot disease, is considered the most destructive disease of citrus at present and threatens citrus pro-duction in all affected areas. Fruit on HLB affected trees remain small, often become misshapen, may develop an undesirable taste, and drop prematurely. Losses to juice orange production in Florida from HLB during pro-duction seasons 2006–07 through 2010–11 were estimated to be above 20% (Hodges and Spreen, 2012). Another effect of HLB is the appearance of yellow shoots in the canopy of diseased trees, which are the result of an asymmetric blotchy mottling of leaves or severe chlorosis, often resembling zinc or other nutritional deficiencies (McClean and Schwarz, 1970). At advanced stages of the disease, twig dieback occurs and trees de-cline. Transmission of Las occurs through the Asian citrus psyllid Diaphorina citri (Halbert, 2005; Halbert and Manjunath, 2004), which feeds on plant phloem. Other modes of transmission are through dodder (Cuscuta sp.) or grafting with infected bud-wood (Halbert and Manjunath, 2004). Two other bacterial pathogens, Ca. L. africanus (Jagoueix et al., 1994) and Ca. L. americanus (Teixeira et al., 2005), are known to cause HLB but are geographically more restricted. HLB affects all known Citrus species and Citrus relatives, and most commercial cultivars
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    • "Although a " nearly complete set of 30 " flagellar biosyn - thetic genes were reported in ' Ca . L . asiaticus ' , some of the flagella biosynthetic genes were reported as pseudogenes ( Duan et al . 2009 ) . However , no ' Ca . L . asiaticus ' or ' Ca . L . ameri - canus ' flagella have been reported to be observed in any publi - cations , despite numerous electron micrographs of these bacte - ria infecting plants and psyllids ( Bove 2006 ) . Nevertheless , one of the ' Ca . L . asiaticus ' genes encodes a predicted flagel"
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    ABSTRACT: Liberibacters form a Rhizobiaceae clade of phloem-limited pathogens of limited host range. Two obligately parasitic species have been sequenced: Candidatus Liberibacter asiaticus (Las), which causes citrus Huanglongbing (HLB) world-wide, and Ca. L. solanacearum (Lso), which causes potato "zebra chip" disease. A third species, Liberibacter crescens (Lcr), was isolated from mountain papaya, grown in axenic culture and sequenced. In an effort to identify common host determinants, the complete genomic DNA sequence of a second HLB species, Ca. L. americanus (Lam) strain "São Paulo" was determined. The circular genome of 1,195,201 bp had an average 31.12% GC content and 983 predicted protein encoding genes, 800 (81.4%) of which had a predicted function. There were 658 genes common to all sequenced liberibacters and only 8 genes common to Lam and Las but not found in Lso. Surprisingly, most of the lipopolysaccharide biosynthetic genes were missing from the Lam genome, as well OmpA and a key regulator of flagellin, all indicating a Lam strategy of avoiding production of major pathogen-associated molecular patterns (PAMPs) present in Las and Lso. As with Las, one of two Lam prophages replicated as an excision plasmid and carried potential lysogenic conversion genes that appeared fragmentary or degenerated in Lso.
    Molecular Plant-Microbe Interactions 02/2014; 27(2):163. DOI:10.1094/MPMI-09-13-0292-R · 4.46 Impact Factor
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