Abstract
Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Developing effective therapies remains a major challenge, as CLas colonizes the host phloem systemically and cannot be cultured in vitro. This study presents a rapid, reproducible, and cost-effective in vivo platform for screening bacteriostatic and bactericidal compounds using CLas-infected citron (Citrus medica (L.) Osbeck) stem cuttings. Among seven citrus genotypes tested, citron stem cuttings exhibited superior rooting performance and uniform vegetative growth. Four propagation protocols were developed and assessed based on the dynamics of rooting and shoot growth, CLas colonization, and the response to oxytetracycline (OTC) treatment. CLas + stem cuttings were treated with OTC via drenching at different developmental root stages. The roots or new vegetative flushes were sampled for bacterial quantification by qPCR. In Protocol 2, in which treatments were applied and sampled 14 and 35 days after planting (DAP) respectively, OTC-treated roots achieved the highest suppression of CLas and a lower incidence of CLas + rooted cuttings compared to non-treated roots. Time-course analysis showed that OTC delayed bacterial establishment in root tissues, with maximal suppression observed at 35 DAP. The proposed protocols simulate the natural progression of systemic infection in citrus plants, allow the assessment of phytotoxicity, and offer a scalable technology that does not underestimate the efficacy of future bactericidal candidates. This platform significantly reduces time and cost compared to traditional seedling or nursery tree experiments and enhances the early-phase screening of antimicrobial compounds. Altogether, this stem cutting-based approach represents a biologically relevant, scalable tool to accelerate therapeutic discovery and strengthen integrated HLB management strategies.
Data availability
The manuscript and supporting files include all data generated during this study.
References
Bové, J. M. Huanglongbing or yellow shoot, a disease of Gondwanan origin: will it destroy citrus worldwide? Phytoparasitica 42, 579–583 (2014).
Bové, J. M. Huanglongbing: A Destructive, Newly-Emerging, Century-Old disease of citrus. Journal Plant. Pathology 88, 7–37 (2006).
Bassanezi, R. B. et al. HLB progress and yield sustainability in mature sweet orange orchards treated with nutritional and elicitor products. PhytoFrontiers™ 4, 213–222 (2024).
Burckhardt, D., Ouvrard, D. & Percy, D. M. An updated classification of the jumping plant-lice (Hemiptera: Psylloidea) integrating molecular and morphological evidence. Eur. J. Taxon. 736, 137–182 (2021).
Ghanim, M. et al. ‘Candidatus Liberibacter asiaticus’ accumulates inside endoplasmic reticulum associated vacuoles in the gut cells of Diaphorina citri. Sci Rep (2017).
Raiol-Junior, L. L., Cifuentes-Arenas, J. C., Cunniffe, N. J., Turgeon, R. & Lopes, S. A. Modeling ‘Candidatus liberibacter asiaticus’ movement within citrus plants. Phytopathology 111, 1711–1719 (2021).
Pandey, S. S., Li, J., Oswalt, C. & Wang, N. Dynamics of ‘Candidatus liberibacter asiaticus’ Growth, concentrations of reactive oxygen Species, and ion leakage in Huanglongbing-Positive sweet orange. Phytopathology 114, 961–970 (2024).
USDA. Citrus summary production, price and value production by county. (2024). https://www.nass.usda.gov/Statistics_by_State/Florida/Publications/Citrus/Citrus_Summary/Citrus_Summary_Prelim/cit082924.pdf (2025).
Graham, J. H., Bassanezi, R. B., Dawson, W. O. & Dantzler, R. Management of Huanglongbing of citrus: lessons from São. Paulo Fla. 32, 34 (2025).
Bassanezi, R. B. et al. Overview of citrus Huanglongbing spread and management strategies in Brazil. Trop. Plant. Pathol. 45, 251–264 (2020).
Fundecitrus. Inventário das laranjeiras da citricultura brasileira. (2025). https://www.fundecitrus.com.br/noticias/safra-de-laranja-202425-e-encerrada-com-producao-total-de-23087-milhoes-de-caixas/
Fundecitrus. Avalia psilídeo. https://app.powerbi.com/view?r=eyJrIjoiNDE1NmFhYzQtZDBmZi00MjQwLWI4ZGEtOTViNTlkZmYzZDIxIiwidCI6ImUzODU0MTM3LWVmMDMtNDU5Ni05M2QwLWIxM2MyMTk4MTU1ZSJ9. (2025).
Albrecht, U., Tardivo, C., Moreno, G. & de Freitas, J. Systemic Delivery of Oxytetracycline by Drill-Based and Drill-Free Trunk Injection for Treatment of Huanglongbing in Young Sweet Orange Trees. Horticulturae 11, (2025).
Ammar, E. D., Shatters, R. G. & Hall, D. G. Localization of Candidatus liberibacter asiaticus, associated with citrus Huanglongbing Disease, in its psyllid vector using fluorescence in situ hybridization. J. Phytopathol. 159, 726–734 (2011).
Hijaz, F. & Killiny, N. Collection and chemical composition of phloem Sap from Citrus sinensis L. Osbeck (sweet orange). PLoS One 9, e101830 (2014).
Attaran, E. et al. Controlled replication of ‘Candidatus liberibacter asiaticus’ DNA in citrus leaf discs. Microb. Biotechnol. 13, 747–759 (2020).
Merfa, M. V., Naranjo, E., Shantharaj, D. & de la Fuente, L. Growth of ‘Candidatus Liberibacter asiaticus’ in Commercial Grapefruit Juice-Based Media Formulations Reveals Common Cell Density-Dependent Transient Behaviors. Phytopathology 112, 131–144 (2022).
Killiny, N., Nehela, Y., One Target, T. & Mechanisms The impact of ‘Candidatus liberibacter asiaticus’ and its Vector, Diaphorina citri, on citrus leaf pigments. Mol. Plant-Microbe Interactions ®. 30, 543–556 (2017).
Leonard, M. T., Fagen, J. R., Davis-Richardson, A. G., Davis, M. J. & Triplett, E. W. Complete genome sequence of Liberibacter crescens BT-1. Stand. Genomic Sci. 7, 271–283 (2012).
Jain, M., Munoz-Bodnar, A., Zhang, S. & Gabriel, D. W. A secreted ‘Candidatus liberibacter asiaticus’ Peroxiredoxin simultaneously suppresses both localized and systemic innate immune responses in planta. Mol. Plant Microbe Interact. 31, 1312–1322 (2018).
Irigoyen, S. et al. Plant hairy roots enable high throughput identification of antimicrobials against Candidatus liberibacter spp. Nat. Commun. 11, 5802 (2020).
Akasenov, A. et al. Spatial chemistry of citrus reveals molecules bactericidal to Candidatus liberibacter Asiaticus. Sci. Rep. 14, 20306 (2024).
Singh, K. K. Propagation of citrus species through cutting: A review. J. Med. Plants Stud. 6, 167–172 (2018).
Debnath, S., Hore, J. K., Dhua, R. S. & Sen, S. K. Auxin synergists in the rooting of stem cutting of lemons (Citrus Limon Burm). South. Indian Hort. 34, 123–128 (1986).
Coletta-Filho, H. D. et al. In planta multiplication and graft transmission of ‘Candidatus liberibacter asiaticus’ revealed by real-time PCR. Eur. J. Plant. Pathol. 126, 53–60 (2010).
Zheng, Z. et al. Unusual five copies and dual forms of NrdB in ‘Candidatus liberibacter asiaticus’: biological implications and PCR detection application. Sci. Rep. 6, 39020 (2016).
Cifuentes-Arenas, J. C., de Goes, A., de Miranda, M. P., Beattie, G. A. C. & Lopes, S. A. Citrus flush shoot ontogeny modulates biotic potential of Diaphorina citri. PLoS One 13, e0190563 (2018).
Archer, L., Kunwar, S., Alferez, F., Batuman, O. & Albrecht, U. Trunk injection of Oxytetracycline for Huanglongbing management in mature grapefruit and sweet orange trees. Phytopathology 113, 1010–1021 (2023).
Harrison, B. P. et al. Controlling HLB with oxytetracycline - Patent Invaio, WO2023/240208 A1 (2023).
Ozias-Akins, P. & Vasil, I. K. Callus induction and growth from the mature embryo of triticum aestivum (Wheat). Protoplasma 115, 104–113 (1983).
Teixeira, D. D. C. et al. Citrus Huanglongbing in São Paulo State, brazil: PCR detection of the ‘Candidatus’ liberibacter species associated with the disease. Mol. Cell. Probes. 19, 173–179 (2005).
Murray, M. G. & Thompson, W. F. Rapid isolation of high molecular weight plant ONA. Nucleic Acids Research 8, 4321–4326 (1980). https://academic.oup.com/nar/article/8/19/4321/2381063
Martins, E. C., Teixeira, D. C., Coletti, D. A. B. & Wulff, N. A. Multiplex quantitative PCR for the detection of bacteria associated with Huanglongbing ‘Candidatus liberibacter asiaticus,’ ‘ Ca. L. americanus,’ and 16Sr IX group Phytoplasma. Plant. Dis. 109, 623–632 (2025).
R Foundation for Statistical Computing. R Core Team. R: A language and environment for statistical computing. Preprint at. (2025).
Younas, M., Zheng, Z., Atiq, M., Wei, T. & Li, Y. Insights into the Huanglongbing pandemic focusing on transmission biology, virulence factors and multifaceted management strategies. Pest Manage. Science Preprint at. https://doi.org/10.1002/ps.70073 (2025).
Chen, X. D. et al. Feeding behavior and hormoligosis associated with Imidacloprid resistance in Asian citrus psyllid, Diaphorina citri. Insect Sci. 31, 1211–1221 (2024).
B Carpenter, J. Occurrence and inheritance of preformed root primordia in stems of Citron (Citrus medica L). Proc. Am. Soc. Hortic. Sci. 77, 211–218 (1961).
Duran-Vila, N., Ortega, V. & Navarro, L. Morphogenesis and tissue cultures of three citrus species. Plant. Cell. Tissue Organ. Cult. 16, 123–133 (1989).
Karp, D. & Hu, X. The Citron (Citrus medica L.) in China. in Horticultural Reviews 143–196 (Wiley, doi:https://doi.org/10.1002/9781119431077.ch5. (2018).
Bowman, K. D. & Joubert, J. Citrus rootstocks. in The Genus Citrus 105–127Elsevier, (2020). https://doi.org/10.1016/B978-0-12-812163-4.00006-1
Ghorbel, R., Navarro, L. & Duran-Vila, N. Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C aurantium) and Alemow (C. macrophylla). J. Hortic. Sci. Biotechnol. 73, 323–327 (1998).
Platt, R. G. and O. K. W. The propagation of citrus. in The Citrus Industry. (ed Reuther, W.) 1–47, (Bekerly: University of California Press, (1973).
Wang, N. & Trivedi, P. Citrus huanglongbing: A newly relevant disease presents unprecedented challenges. Phytopathology 103, 652–665 (2013).
Hu, J. & Wang, N. Evaluation of the Spatiotemporal dynamics of Oxytetracycline and its control effect against citrus Huanglongbing via trunk injection. Phytopathology 106, 1495–1503 (2016).
Vincent, C. I., Hijaz, F., Pierre, M. & Killiny, N. Systemic uptake of Oxytetracycline and streptomycin in Huanglongbing-Affected citrus groves after foliar application and trunk injection. Antibiotics 11, 1092 (2022).
Batuman, O. et al. The use and impact of antibiotics in plant agriculture: A review. Phytopathology 114, 885–909 (2024).
Lopes, S. A. & Cifuentes-Arenas, J. C. Protocol for successful transmission of ‘Candidatus liberibacter asiaticus’ from citrus to citrus using Diaphorina citri. Phytopathology 111, 2367–2374 (2021).
Pang, Z. et al. Citrus CsACD2 is a target of Candidatus liberibacter Asiaticus in Huanglongbing disease. Plant. Physiol. 184, 792–805 (2020).
Huang, C. Y. et al. A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing. Proceedings of the National Academy of Sciences 118, (2021).
Rao, W. et al. ZnO quantum Dots reduce acquisition of Huanglongbing pathogen by Diaphorina citri. Pest Manag Sci. 81, 2712–2721 (2025).
Bendix, C. & Lewis, J. D. The enemy within: phloem-limited pathogens. Mol. Plant. Pathol. 19, 238–254 (2018).
Kruse, A., Fleites, L. A. & Heck, M. Lessons from one fastidious bacterium to another: what can we learn about liberibacter species from xylella fastidiosa. Insects 10, 300 (2019).
Pandey, S. S., Hendrich, C., Andrade, M. O. & Wang, N. Candidatus Liberibacter: From Movement, Host Responses, to Symptom Development of Citrus Huanglongbing. Phytopathology 112, 55–68 (2022).
Pandey, S. S., Vasconcelos, N. C., Wang, N. & F. & Spatiotemporal dynamics of ‘Candidatus liberibacter asiaticus’ colonization inside citrus plant and Huanglongbing disease development. Phytopathology 111, 921–928 (2021).
Wei, H., Tang, M. & Xu, X. Mechanism of uptake, accumulation, transport, metabolism and phytotoxic effects of pharmaceuticals and personal care products within plants: A review. Sci. Total Environ. 892, 164413 (2023).
Killiny, N. et al. Tracing penicillin movement in citrus plants using Fluorescence-Labeled penicillin. Antibiotics 8, 262 (2019).
Enstone, D. E., Peterson, C. A. & Ma, F. Root endodermis and exodermis: Structure, Function, and responses to the environment. J. Plant. Growth Regul. 21, 335–351 (2002).
Péret, B., Larrieu, A. & Bennett, M. J. Lateral root emergence: a difficult birth. J. Exp. Bot. 60, 3637–3643 (2009).
Higgins, S. A. et al. Plant-Derived, Nodule-Specific Cysteine-Rich peptides as a novel source of biopesticides for controlling citrus greening disease. Phytopathology 114, 971–981 (2024).
Johnson, E. G., Wu, J., Bright, D. B. & Graham, J. H. Association of ‘Candidatus liberibacter asiaticus’ root infection, but not phloem plugging with root loss on huanglongbing-affected trees prior to appearance of foliar symptoms. Plant. Pathol. 63, 290–298 (2014).
Blaustein, R. A., Lorca, G. L. & Teplitski, M. Challenges for managing Candidatus liberibacter spp. (Huanglongbing disease Pathogen): current control measures and future directions. Phytopathology 108, 424–435 (2018).
Zhang, M. et al. Screening molecules for control of citrus Huanglongbing using an optimized regeneration system for Candidatus liberibacter Asiaticus infected periwinkle (Catharanthus roseus) cuttings. Phytopathology 100, 239–245 (2010).
Rhodes, B. H. et al. Direct Infusion Device for Molecule Delivery in Plants. Journal of Visualized Experiments (2023). (2023).
Kennedy, J. P. et al. A perspective on current therapeutic molecule screening methods against ‘Candidatus liberibacter asiaticus’, the presumed causative agent of citrus Huanglongbing. Phytopathology 113, 1171–1179 (2023).
Louzada, E., Vazquez, O., Chavez, S., Sétamou, M. & Kunta, M. Optimization of VqPCR for reliable detection of viable candidatus liberibacter Asiaticus in citrus. HortScience 57, 692–697 (2022).
Acknowledgements
The authors thank Elaine C. Martins for sharing the quantification curve data for CLas titer estimation, and Isabela V. Primiano for the contribution to Figure 2.
Funding
B. C. P. S. received a grant from Fundecitrus ID: 04 3119. N. A. W. has a CNPq fellowship (306365/2024-3).
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B.C.P.S. and F.B. designed research. E.S.G. analyzed data. B.C.P.S., T.A.S. performed research. N.A.W. provided *C. medica* plants and reagents. B.C.P.S wrote the manuscript. All authors reviewed and approved the final version of the manuscript.
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Pecoraro Sanches, B.C., dos Santos, T.A., Gorayeb, E.S. et al. A Citrus medica stem cutting-based platform for rapid screening of therapeutic compounds against Candidatus Liberibacter asiaticus. Sci Rep (2026). https://doi.org/10.1038/s41598-026-37186-7
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DOI: https://doi.org/10.1038/s41598-026-37186-7
