An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus

Authors: KRYSTEL, JOSEPH; SHI, QINGCHUN; Shaw, Jefferson; GOUTAM, GUPTA - NEW MEXICO CONSORTIUM; HALL, DAVID; STOVER, EDDIE

Submitted to: Plant Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2019
Publication Date: 7/31/2019
Citation: Krystel, J.A., Shi, Q., Shaw, J.J., Goutam, G., Hall, D.G., Stover, E.W. 2019. An in vitro protocol for rapidly assessing the effects of antimicrobial compounds on the unculturable bacterial plant pathogen, Candidatus Liberibacter asiaticus. Plant Methods. 15:85. https://doi.org/10.1186/s13007-019-0465-1
DOI: https://doi.org/10.1186/s13007-019-0465-1

Interpretive Summary: Research into Huanglongbing (Greening) disease in citrus is severely limited due to the unculturable nature of the causal pathogen, Candidatus Liberibacter asiaticus (CLas). We have developed an in vitro assay to test potential CLas-killing therapeutic agents that provides a substitute for more traditional culture based assays. This assay uses viable CLas cells isolated in a homogenate of the insect vector Diaphorina citri and represents a significant savings in cost and time when compared to other available methods.

Technical Abstract: Candidatus Liberibacter asiaticus (CLas) is the most impactful pathogen on citrus production, is vectored by the Asian citrus psyllid (ACP, Diaphorina citri), and lacks effective treatment or resistant cultivars. Research into CLas pathogenicity and therapy has been hindered by the lack of persistent pure cultures. Work to date has been mostly limited to in planta studies that are time and resource intensive. We developed and optimized an in vitro protocol to quickly test the effectiveness of potential therapeutic agents against CLas. The assay uses intact bacterial cells contained in homogenized CLas-infected ACP tissue and a propidium monoazide (PMA) viability assay to measure antimicrobial activity. The applicability of PMA was evaluated; with the ability to differentiate between viable and non-viable CLas cells confirmed using multiple bactericidal treatments. We identified light activation conditions to prevent Polymerase chain reaction (PCR) interference and identified a suitable positive control for nearly complete CLas disruption (0.1% triton-x 100). The mature protocol was used to compare a panel of peptides already under study for potential CLas targeting bactericidal activity. One of the peptides, CysP26, showed especially marked ability to lyse CLas cells in this assay; a result consistent with previous in silico and in vitro studies. This psyllid homogenate assay allows for a quick and accurate assessment of potential CLas-disrupting peptides. Comparison within a uniform isolate largely eliminates experimental error arising from variation in CLas titer between and within individual host organisms, and using a viability assay permits direct assessment of potential therapeutic compounds without generating pure cultures or conducting extensive in planta or field studies.