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.

SUMMARYTaxonomy: Bacteria; Proteobacteria, Gammaproteobacteria; Xanthomonadales; Xanthomonadaceae, Xanthomonas.Microbiological properties: Gram-negative, obligately aerobic, straight rods, motile by a single polar flagellum, yellow pigment.Related species: X. campestris , X. axonopodis , X. oryzae , X. albilineans .Host range: Affects Rutaceous plants, primarily Citrus spp., Fortunella spp., and Poncirus spp., world-wide. Quarantined pathogen in many countries. Economically important hosts are cultivated orange, grapefruit, lime, lemon, pomelo and citrus rootstock.Disease symptoms: On leaves, first appearance is as oily looking, 2-10 mm, similarly sized, circular spots, usually on the abaxial surface. On leaves, stems, thorns and fruit, circular lesions become raised and blister-like, growing into white or yellow spongy pustules. These pustules then darken and thicken into a light tan to brown corky canker, which is rough to the touch. On stems, pustules may coalesce to split the epidermis along the stem length, and occasionally girdling of young stems may occur. Older lesions on leaves and fruit tend to have more elevated margins and are at times surrounded by a yellow chlorotic halo (that may disappear) and a sunken centre. Sunken craters are especially noticeable on fruit, but the lesions do not penetrate far into the rind. Defoliation and premature abscission of affected fruit occurs on heavily infected trees.

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.

A recent epiphytotic disease on citrus in Florida nurseries was caused by strains of Xanthomonas campestris with different host specificity and lower pathogenic capacities than those of previously described strains of X. campestris pv. citri. The new strains were classified as X. campestris pv. citri because they were isolated from rutaceous hosts and despite the fact that they caused a different disease than strains previously described in that pathovar. Restriction fragment length polymorphism analyses revealed that the Florida strains comprised a heterogeneous (E) group, interrelated with X. campestris pv. alfalfae, X . campestris pv. cyamopsidis, and X. campestris pv. dieffenbachiae. In contrast, the previously described strains of X. campestris pv. citri formed two highly distinct, homogeneous (A and B/C/D) groups. Furthermore, the strains of X. campestris pv. campestris, X . campestris pv. glycines, X . campestris pv. malvacearum, X . campestris pv. phaseoli, X . campestris pv. pisi, and X. campestris pv. vignicola tested were also distinctive and appeared to be only distantly related to one another and to all X. campestris pv. citri strains. We concluded (i) that some pathovars are sufficiently distinct from the type strain of the species X. campestris to be considered as separate species, (ii) that X. campestris pv. citri group A and X. campestris pv. phaseoli (not including X . campestris pv. phaseoli var. fuscans) represent distinctly separate subbranches of the genus and should be respectively reinstated to species as X. citri (ex Hasse) nom. rev. 3213 and X. phaseoli (ex Smith) nom. rev. 627, and (iii) that the X. campestris pv. citri B/C/D strains and the heterogeneous E strains should be respectively renamed X. campestris pv. aurantifolii pv. nov. and X. campestris pv. citrumelo pv. nov.

'Candidatus Liberibacter asiaticus' is transmitted by psyllids and causes huanglongbing (HLB), a lethal disease of citrus. Most pathogenic 'Ca. L. asiaticus' strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high-copy excision plasmid encoding a reactive oxygen species-scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus and was suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into Escherichia coli and Liberibacter crescens, a culturable proxy for 'Ca. L. asiaticus'. Transformed L. crescens cells showed 20 to 25% enhanced resistance to H₂O₂on agar plates, 47% greater enzymatic activity, and enhanced growth in liquid cultures. A nonclassical secretion potential was predicted for SC2_gp095 and secretion from L. crescens was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta resulted in strong transcriptional downregulation of RbohB, the key gatekeeper of the H₂O₂-mediated defense signaling in plants, helping explain the surprisingly long incubation period (years) before HLB symptoms appear in 'Ca. L. asiaticus'-infected citrus. 'Ca. L. asiaticus' peroxidase is likely a secreted, horizontally acquired effector that suppresses host symptom development, a tactic used by most biotrophic plant pathogens.