Zhou, Lijuan

‘Candidatus Liberibacter asiaticus’ (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

ABSTRACT “ Candidatus Liberibacter asiaticus” is a psyllid-transmitted, phloem-limited alphaproteobacterium and the most prevalent species of “ Ca . Liberibacter” associated with a devastating worldwide citrus disease known as huanglongbing (HLB). Two related and hypervariable genes ( hyv I and hyv II ) were identified in the prophage regions of the Psy62 “ Ca. Liberibacter asiaticus” genome. Sequence analyses of the hyv I and hyv II genes in 35 “ Ca. Liberibacter asiaticus” DNA isolates collected globally revealed that the hyv I gene contains up to 12 nearly identical tandem repeats (NITRs, 132 bp) and 4 partial repeats, while hyv II contains up to 2 NITRs and 4 partial repeats and shares homology with hyv I . Frequent deletions or insertions of these repeats within the hyv I and hyv II genes were observed, none of which disrupted the open reading frames. Sequence conservation within the individual repeats but an extensive variation in repeat numbers, rearrangement, and the sequences flanking the repeat region indicate the diversity and plasticity of “ Ca. Liberibacter asiaticus” bacterial populations in the world. These differences were found not only in samples of distinct geographical origins but also in samples from a single origin and even from a single “ Ca. Liberibacter asiaticus”-infected sample. This is the first evidence of different “ Ca. Liberibacter asiaticus” populations coexisting in a single HLB-affected sample. The Florida “ Ca. Liberibacter asiaticus” isolates contain both hyv I and hyv II , while all other global “ Ca. Liberibacter asiaticus” isolates contain either one or the other. Interclade assignments of the putative Hyv I and Hyv II proteins from Florida isolates with other global isolates in phylogenetic trees imply multiple “ Ca. Liberibacter asiaticus” populations in the world and a multisource introduction of the “ Ca. Liberibacter asiaticus” bacterium into Florida.