Li, Wenbin

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.

Citrus huanglongbing (HLB), or greening disease, is strongly associated with any of three nonculturable gram-negative bacteria belonging to ‘Candidatus Liberibacter spp.’ ‘Ca. Liberibacter spp.’ are transmitted by citrus psyllids to all commercial cultivars of citrus. The diseases can be lethal to citrus and have recently become widespread in both São Paulo, Brazil, and Florida, United States, the locations of the largest citrus industries in the world. Asiatic HLB, the form of the disease found in Florida, is associated with ‘Ca. Liberibacter asiaticus’ and is the subject of this report. The nonculturable nature of the pathogen has hampered research and little is known about the distribution of ‘Ca. L. asiaticus’ in infected trees. In this study, we have used a quantitative polymerase chain reaction assay to systematically quantify the distribution of ‘Ca. L. asiaticus’ genomes in tissues of six species of citrus either identified in the field during survey efforts in Florida or propagated in a greenhouse in Beltsville, MD. The populations of ‘Ca. L. asiaticus’ inferred from the distribution of 16S rDNA sequences specific for ‘Ca. L. asiaticus’ in leaf midribs, leaf blades, and bark samples varied by a factor of 1,000 among samples prepared from the six citrus species tested and by a factor of 100 between two sweet orange trees tested. In naturally infected trees, above-ground portions of the tree averaged 1010 ‘Ca. L. asiaticus’ genomes per gram of tissue. Similar levels of ‘Ca. L. asiaticus’ genomes were observed in some but not all root samples from the same plants. In samples taken from greenhouse-inoculated trees, levels of ‘Ca. L. asiaticus’ genomes varied systematically from 104 genomes/g at the graft inoculation site to 1010 genomes/g in some leaf petioles. Root samples from these trees also contained ‘Ca. L. asiaticus’ at 107 genomes/g. In symptomatic fruit tissues, ‘Ca. L. asiaticus’ genomes were also readily detected and quantified. The highest levels of ‘Ca. L. asiaticus’ in fruit tissues were found in the locular membranes and septa (108 genomes/g), with 100-fold lower levels of ‘Ca. L. asiaticus’ in the meso and pericarp of such fruit. Our results demonstrate both the ubiquitous presence of ‘Ca. L. asiaticus’ in symptomatic citrus trees as well as great variation between individual trees and among samples of different tissues from the same trees. Our methods will be useful in both the management and scientific study of citrus HLB, also known as citrus greening disease.

Citrus huanglongbing (HLB), also known as citrus greening or citrus yellow shoot, is considered the most serious disease of citrus worldwide. The disease has Asian, African, and American forms caused by “Candidatus Liberibacter asiaticus”, “Ca. L. africanus”, and “Ca. L. americanus”, respectively, which can be spread efficiently by the psyllid vectors Diaphorina citri and Trioza erytreae and through contaminated plant materials. Infected citrus groves are usually destroyed or become unproductive in 5 to 8 years. The presumed low concentration and uneven distribution of the pathogens in citrus plants and vector insects make the phloem-limited bacterium difficult to detect consistently. In this study, we compared and validated four conventional polymerase chain reaction (PCR)-based protocols, one loop-mediated isothermal amplification (LAMP) protocol, and three TaqMan real-time PCR protocols. The detection sensitivity of the validated conventional PCR assays reported are improved compared with the original protocols. All of the validated conventional and the newly developed real-time methods were reliable for confirmatory tests for the presence of “Ca. Liberibacter spp.” in symptomatic samples. There were no differences in assay specificity among the standard format PCR-based methods evaluated. The TaqMan real-time PCR was 10- to 100-fold more sensitive than conventional PCR and LAMP, showing the potential to become a valuable tool for early detection and identification of “Ca. Liberibacter spp.” prior to the appearance of disease symptoms. The methods validated in this study will be very useful for regulatory response, effective management of infected trees, and development of a “Ca. Liberibacter spp.”-free nursery system.