A Perspective on Current Therapeutic Molecule Screening Methods Against ‘Candidatus Liberibacter asiaticus’, the Presumed Causative Agent of Citrus Huanglongbing

Abstract

Huanglongbing (HLB), referred to as citrus greening disease, is a bacterial disease impacting citrus production worldwide and fatal in young trees and certain varieties. In some areas, the disease is devastating the citrus industry. A successful solution to HLB will be measured in economics: Citrus growers need treatments that improve tree health, fruit production, and most importantly economic yield. The profitability of citrus groves is the ultimate metric that truly matters when searching for solutions to HLB. Scientific approaches used in the laboratory, greenhouse, or field trials are critical to discovery of those solutions and to estimate the likelihood of success of a treatment aimed for commercialization. Researchers and the citrus industry use a number of proxy evaluations of potential HLB solutions and understanding the strengths and limitations of each assay as well as how best to compare different assays is critical for decision making to advance therapies into field trials and commercialization. This perspective aims to help the reader compare and understand the limitations of different proxy evaluation systems based on the treatment and evaluation under consideration. The researcher must determine the suitability of one or more of these metrics to identify treatments and predict the usefulness of these treatments in having an eventual impact on citrus production and HLB mitigation. As therapies advance to field trials in the next few years, a re-evaluation of these metrics will be useful to guide future research efforts on strategies to mitigate HLB and vascular bacterial pathogens in other perennial crops.

Spatial chemistry of citrus reveals molecules bactericidal toCandidatusLiberibacter asiaticus

“…To confirm that these molecules are indeed toxic to C Las, we conducted the disc and hairy roots assays that confirmed their bactericidal effects toward C Las. Although the disc assay exploited a surrogate organism, Liberibacter crescens, the hairy roots assay is a way to directly assess the toxicity, both genetic- and chemical-based to C Las itself 28,37,38 . In this approach, C Las is maintained in citrus hairy root tissues, and the antimicrobials are directly infiltrated into the roots by vacuum, thus overcoming the delivery bottleneck that exists with whole tree assays 7,29,39 and circumventing concomitant irreproducibility issues.…”

Plant-derived, nodule-specific cysteine rich peptides inhibit growth and psyllid acquisition of ‘CandidatusLiberibacter asiaticus’, the citrus Huanglongbing bacterium

“…relatives are unculturable. It is not practical to screen a large number of antimicrobial compounds against CLas in leaves or insects (Kennedy et al 2023). Therefore, we used the culturable relative of CLas, L. crescens, as a surrogate for CLas in bacterial culture assays, which can be carried out in 96-well plates, a more high-throughput way to narrow down the more than 600 NCR candidates to high priority candidates for testing against CLas in more laborious assays.…”

“…These are two required properties for antimicrobial peptides effective as HLB therapies. While the use of other powerful CLas screening systems have been reported, such as the hairy root system (Irigoyen et al 2020;Mandadi et al 2020), only assays that incorporate vascular mobility (movement in xylem or phloem) as a component of the screen, such as the excised leaf assay, can ascertain whether an antimicrobial peptide is mobile and stable in the citrus plant's vascular system (Kennedy et al 2023). In our experiments, we evaluated both CLas DNA and RNA since some antimicrobials may induce dormancy or initial death of the bacterium without rapid induction of cell lysis and DNA degradation.…”

“…Alternatively, it is possible that the NCR peptides interfered with CLas acquisition by psyllid nymphs due to induction of changes in CLas physiology or morphotype in the plant and not necessarily by killing CLas, so populations of CLas could remain viable and rebounded despite being unable to be acquired by psyllids. Differentiating between live and dead CLas extracted from plant tissue is not reliable (reviewed by (Kennedy et al 2023), so this was not attempted. NCR peptides have been shown to regulate the morphotype of S. meliloti (Montiel et al 2017), including induction of biofilm formation and production of extracellular vesicles.…”

“…The bacteria replicate to high levels in the psyllid salivary glands by two weeks after adult emergence ). Thus, new antimicrobial therapies that are phloem-mobile and can block CLas acquisition by nymphs are paramount for the control of HLB (Kennedy et al 2023).…”