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Strain tracking of 'Candidatus Liberibacter asiaticus', citrus greening disease pathogen, enabled by high-resolution microbiome analysis of the Asian citrus psyllid.

The Asian citrus psyllid, Diaphorina citri, is an invasive insect and a vector of 'Candidatus Liberibacter asiaticus' (CLas), a bacterium whose growth in Citrus species results in huanglongbing (HLB), also known as citrus greening disease. Methods to enrich and sequence CLas from D. citri often rely on biased genome amplification and nevertheless contain significant quantities of host DNA. To overcome these hurdles, we developed a simple pre-treatment DNase and filtration (hereafter PDF) protocol to remove host DNA and directly sequence CLas and the complete, primarily uncultivable, microbiome from D. citri adults. The PDF protocol yielded CLas abundances upwards of 60% and facilitated direct measurement of CLas and endosymbiont replication rates in psyllids. The PDF protocol confirmed our strains derived from a progenitor Florida CLas strain and accumulated 156 genetic variants, underscoring the utility of this data for bacterial strain tracking. CLas genetic polymorphisms arising in lab-reared psyllid populations included prophage encoding regions with key functions in CLas pathogenesis, putative antibiotic resistance loci, and a single secreted effector. These variants suggest laboratory propagation of CLas may result in different phenotypic trajectories among laboratories, and may confound CLas physiology or therapeutic design and evaluation if these differences remain undocumented. Finally, we obtained genetic signatures affiliated with Citrus nuclear and organellar genomes, entomopathogenic fungal mitochondria, and commensal bacteria from laboratory-reared and field-collected D. citri adults. Hence, the PDF protocol can directly inform agricultural management strategies related to bacterial strain tracking, insect microbiome surveillance, and antibiotic resistance screening.