Aeroponic propagation of citrus trees infected with ‘Candidatus Liberibacter asiaticus’

Authors: SCANLON, LISA - Cornell University; Deblasio, Stacy; SULLIVAN, SAMANTHA - Oak Ridge Institute For Science And Education (ORISE); IGWE, DAVID - Cornell University; THOMAS, CHAD - Cornell University; Heck, Michelle

Submitted to: Protocols.io
Publication Type: Research Technical Update
Publication Acceptance Date: 9/8/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Candidatus Liberibacter asiaticus (CLas), the presumed causal agent of citrus greening disease (also known as Huanglongbing, HLB), remains unculturable under laboratory conditions, severely limiting experimental throughput and the ability to directly interrogate CLas biology. This constraint makes the development of reliable, tractable, reproducible citrus-based systems essential for advancing research on host–pathogen–vector interactions. This protocol describes the vegetative propagation of CLas-infected citrus material using an aeroponic cloning apparatus (EZ-Clone Pro Low Cloning System). By propagating cuttings from flowering, infected trees, researchers can rapidly generate uniform, CLas-positive citrus material for experimental assays. Unlike seed-based propagation, which can take more than a year to generate seedlings with usable stem diameters and up to five years to achieve flowering, this cloning method produces mature, infected plant material in a matter of months. The approach significantly increases research efficiency by providing genetically uniform, CLas-infected clones that retain the maturity of the donor plant. This allows for consistent and accelerated bioassays that are not possible with variable seed-derived plants. The protocol has demonstrated high survival rates across multiple citrus cultivars, with citron clones reaching up to 95% success regardless of cutting diameter. By overcoming the barriers of long generation times and the unculturable nature of CLas, this method provides a critical enabling technology for citrus researchers. It accelerates the pace of experimentation in HLB research, supporting efforts to better understand disease biology and to evaluate management strategies under controlled laboratory and greenhouse conditions.

Technical Abstract: This is a general protocol for the vegetative propagation of citrus material infected with the bacterium Candidatus Liberibacter asiaticus (CLas), the presumed causal agent of citrus greening disease (HLB), using an aeroponic cloning apparatus. The following procedure details a workflow using an EZ-Clone Pro Low Cloning System from Hydrobuilder.com. This system comes in varying cell-sizes (number of clones). We have found the most versatile to be the 16- or 32-cell size based on ease of preparation, keeping algae growth to a minimum, and maintenance. Cloning versus seed sowing has been useful in producing infected material for experimental assays requiring cloned, mature citrus trees as opposed to genetically variable plants from seeds. Where seed germination can take up to 12+ months to achieve plants with stem diameters = 10 mm and 5+ years for flowering, infected branches from flowering trees can be directly propagated using this protocol within a few months and retain their maturity. Clone viability rate differs by citrus cultivar and general health of the cutting used, with citron and CLas negative material having the highest success rate. Infected citron cuttings have had a 95% survival rate, regardless of the diameter of the cutting. The cutting diameter ranged from 2mm-12mm and was limited to 12mm maximum, which is the size of the collar on the EZ-Clone Pro Low cloner.