Strain tracking of ‘candidatus liberibacter asiaticus’, citrus greening disease pathogen, enabled by high-resolution microbiome analysis of the Asian citrus psyllid

Authors: HIGGINS, STEVEN - Former ARS Employee; MANN, MARINA - Cornell University; Heck, Michelle

Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/9/2022
Publication Date: 6/9/2022
Citation: Higgins, S., Mann, M., Heck, M.L. 2022. Strain tracking of ‘candidatus liberibacter asiaticus’, citrus greening disease pathogen, enabled by high-resolution microbiome analysis of the Asian citrus psyllid. Phytopathology. 112(11):2273-2287. https://doi.org/10.1094/PHYTO-02-22-0067-R.
DOI: https://doi.org/10.1094/PHYTO-02-22-0067-R

Interpretive Summary: Citrus greening disease is the most serious disease of citrus. In the US, the disease is endemic in Florida and spreading in California and Texas. Citrus greening disease is ultimately fatal for infected citrus trees, and there is no cure. The disease is associated with plant infection by a bacterium, referred to as CLas. CLas is spread by the Asian citrus psyllid, a tiny insect the size of a pencil tip, that spreads the bacteria during feeding on infected citrus trees. CLas cannot be cultured in the laboratory so experiments to study how different CLas strains evolve and the development of antibiotic resistant CLas strains have been challenging. In this work, we developed a method to enrich CLas cells from single psyllid insects for genome sequencing. The method, referred to as the PDF method, enabled scientists to enrich CLas cells and greatly reduce the number of psyllid cells when preparing samples for genome sequencing. Using the PDF method, we have generated genome sequence datasets with CLas relative abundances upwards of 60%. This allows us to track the precise strain of CLas from a single insect, which may inform the origin of new outbreaks. Furthermore, we also detected and analyzed data from other bacterial populations present within psyllids processed using the PDF protocol, suggesting it is a comprehensive method to simultaneously explore multiple insect-associated bacterial populations. The results from application of the PDF method will directly inform agricultural management strategies related to surveillance of insect vectors, insect, animal and plant pathogens, screening for antibiotic resistance, or insect dietary analysis. These results will be of broad interest to the plant pathology and entomology fields as well as important agricultural and human health stakeholders in the US and across the world.

Technical Abstract: 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.