Reduction of Candidatus Liberibacter asiaticus titres in citrus seedlings via root-drenching application of bio-based nanoemulsions

Huanglongbing (HLB) is one of the most devastating diseases of citrus nowadays. The etiological agent is a fastidious and non-culturable bacterium known as Candidatus Liberibacter asiaticus (C Las). C Las is a phloem-restricted pathogen, which makes the efficient delivery of antimicrobial compounds to control it a major challenge for citriculture. Therefore, the development of formulations capable of penetrating plant barriers and reducing the bacterial population within the phloem is critical for the control of HLB. Here we report on the development of a novel formulation loaded with oregano essential oil, hexyl gallate and eugenol, described in the literature as a transdermal permeation enhancer. The most effective formula was selected in vitro against bacterial surrogates (Rhizobium tropici and Rhizobium phaseoli), displaying bactericidal concentrations of 0.25 and 0.5% (v/v), respectively, of bactericidal concentration. The selected formulation was characterized as a near-Newtonian fluid, with 98.5% of particles measuring around 50 nm, a Polydispersity Index of 0.31, and zeta potential of -17.5 mV. Furthermore, Scanning electron microscopy revealed the presence of nanoparticles, alongside microscopic aggregates. The formulation maintained its bactericidal activity over 120-day storage period. During the root-drenching assays, the formulation significantly reduced overall C Las titres by 1.29 and 2.46 log 10 over a period of 300 days, in two independent experiments. Furthermore, ecotoxicological assessments revealed no acute toxicity in Tenebrio molitor or significant effects in the survival of Melipona scutellaris under the tested conditions. Our findings demonstrate the potential of this non-synthetic formulation as an effective and safe alternative for HLB management, without disrupting non-target organisms.

Citrus production is one of the most economically important agricultural sectors worldwide, supporting both fresh fruit markets and the juice industry (Neves et al., 2014). However, this sector faces a major threat known as Huanglongbing (HLB), widely recognized as the most destructive citrus disease globally (Bové, 2006; Gottwald, 2010). The disease has rapidly spread to major citrus-producing regions, leading to significant losses. In Florida (USA), HLB was the main reason for a 90% reduction in citrus production, causing an economic impact that exceeded US$ 4.5 billion (Blaustein et al., 2018; Albrecht et al., 2025; Graham et al., 2024). As the world leader sweet orange producer, Brazil is currently facing a similar epidemiological crisis.

While citrus production reached over 12.5 million tons in 2024, generating approximately 200,000 jobs (FUNDECITRUS, 2024), HLB poses a significant threat to the sustainability of the sector. Since its introduction into Brazil, the disease has caused severe economic and productive losses, including the eradication of approximately 18 million citrus trees between 2004 and 2012 and increases of nearly 15% in citrus production costs associated with disease management and vector control (Bassanezi et al., 2020; De Miranda et al., 2012). Recent surveys from the Brazilian citrus belt demonstrated that greening incidence reached 47.63% of orange trees in 2025, corresponding to nearly 100 million infected trees in the main citrus-producing regions of São Paulo and Minas Gerais states (FUNDECITRUS, 2025).

HLB is associated with phloem-limited bacteria of the genus Candidatus Liberibacter, particularly Candidatus Liberibacter asiaticus (C Las), the most aggressive and widespread species worldwide (Ghosh et al., 2022). The pathogen colonizes sieve elements, disrupting photoassimilate transport, inducing callose deposition, and causing metabolic imbalances that lead to progressive plant decline (Casteel et al., 2012; Li et al., 2017). Infected trees exhibit symptoms such as blotchy mottle, leaf chlorosis, reduced fruit quality, and eventual death, severely impacting productivity (Bové, 2006). The disease is primarily transmitted by the psyllid Diaphorina citri (Kuwayama), a highly efficient vector that acquires and transmits the pathogen, especially during its nymphal stages (Inoue et al., 2009; Ammar El et al., 2016).

Despite the adoption of integrated management strategies, including the use of disease-free seedlings, vector control, and removal of symptomatic plants, HLB remains difficult to control due to the fact that C Las is restricted to the phloem of the host plant (Ghosh et al., 2022). Consequently, alternative approaches have been explored, particularly the use of antimicrobial compounds combined with advanced delivery systems. However, the extensive agricultural use of broad-spectrum antibiotics, such as oxytetracycline, raises environmental concerns, including off target phytotoxicity and the emergence of bacterial resistance (De Gracia Coquerel et al., 2023), prompting the ongoing development of more sustainable alternatives. Several alternative strategies have been explored to the reliance on these broad-spectrum antibiotics. Thermotherapy has shown efficacy in reducing C Las titre, but the technique requires specialized heating equipment, and is a labor and energy intensive method, which could limit its scalability in large commercial orchards (Yang et al., 2016). Recent studies identified four active prophages in the C Las genome, which if activated, could potentially induce bacterial lysis (Ying et al., 2025). However, the unculturable nature of the bacteria make direct studies unfeasible, and this approach faces the challenge of reaching the bacterial target in the plant tissue and keeping the active molecule protected from environmental effects (e.g., UV incidence) (Jain et al., 2017). Antimicrobial peptides, while promising in vitro, are often limited by high production costs and susceptibility to proteolytic degradation under field conditions (Huang et al., 2021). These constraints reveal the need for delivery strategies that combine applicability, cost-effectiveness, and stability.

Nano- and microformulations have emerged as a promising strategy to enhance the efficacy of antimicrobial agents by improving their stability, bioavailability, plant tissue penetration, and transport to vascular tissues (Vatcharakajon et al., 2023; Wang et al., 2022; Yakoubi et al., 2021; Yang et al., 2015). Therefore, the rational development of next-generation antimicrobial formulations guided by the European Union’s Safe-and-Sustainable-by-Design (SSbD) framework emerges as a compelling avenue for HLB control. By incorporating plant-derived bioactive molecules, prioritizing reduced environmental burden, non-target safety, and functional efficacy, SSbD-oriented formulations offer a holistic alternative to conventional antibiotic-based interventions. In this study, we investigated the antimicrobial efficacy, physicochemical stability, and in planta performance of novel formulations targeting C Las, employing surrogate bacterial systems (Duan et al., 2009) and citrus seedlings to assess both biological activity and translational potential. In addition, an initial ecotoxicological assessment was performed using Tenebrio molitor larvae and Melipona scutellaris to evaluate acute mortality and survival following topical exposure, respectively.

Bacterial strains and growth conditions

Due to the unculturable nature of Candidatus Liberibacter asiaticus, direct in vitro screening was not feasible. To overcome this, Rhizobium tropici (R. tropici) (CIAT 899, EMBRAPA: BR322) and Rhizobium phaseoli (R. phaseoli) (EMBRAPA: BR10052), obtained from the Rhizobiales strain collection of Embrapa Seropédica (EMBRAPA, Rio de Janeiro, Brazil) were selected as culturable biological surrogates. These bacteria are of the same order (Rhizobiales) as C Las and share similar genetic and physiological traits (Duan et al., 2009; Hartung et al., 2011). While not directly assessing antibacterial activity in C Las, the use of these surrogates allowed for a rapid, reproducible, and standardized screening platform for initial assessment of formulations. The Lysogeny Broth (LB) was used for bacterial growth and testing (10 g·L - ¹ tryptone, 5 g·L - ¹ yeast extract, and 10 g·L - ¹ NaCl), with the addition of 15 g·L - ¹ agar for solid medium when required. Bacteria were kept at 29 °C, and liquid cultures were incubated under agitation of 180 rpm.

Composition and manufacturing of formulations

Based on previous studies by our group, nine formulations were prepared at the N&P lab, a pharmaceutical technology laboratory, using natural-based components, including cationic polymers, soy lecithin, surfactants, emulsifiers, essential oils, vegetable oils, and micro and macronutrients.

The formulations differ primarily in the proportions and combinations of these components, which directly influence their physicochemical properties and biological activity. The active ingredients, such as essential oils, hexyl gallates, and isolated natural compounds were tested for antibacterial activity against other Gram-negative bacteria, in a diverse array of applications, in previous publications from our group (Battagin et al., 2021; Silva et al., 2013; Cavalca et al., 2020; Zamuner et al., 2023; Braun et al., 2023). Additionally, different techniques were employed for micro and nanoparticle formation, combined with both low-energy homogenization (mechanical stirring at 500–600 rpm) and high-energy homogenization (Ultra-Turrax disperser, IKA, Staufen, Germany, at 15,000 rpm for 5 min).

Antibacterial activity against surrogates

The inhibitory concentrations of the formulations against R. tropici and R. phaseoli were determined through the resazurin microtitre assay (REMA) (Silva et al., 2013). The formulations to be tested were diluted in LB media in a 96-well plate, ranging from 4% to 0.03%, (v/v), in a total volume of 100 µL. As a positive control (PC), the antibiotic Oxytetracycline (0.625 mg.L -1) was used for both bacteria, and for the negative control (NC) we used LB medium only. The bacterial inoculum was standardized to 10 5 cells per well, for both bacteria. After assembling the test plates, bacteria were exposed to the formula for 16 h at 29 °C without agitation. Following incubation, aliquots of each well were transferred to solid LB medium plates, which were further incubated for 48 h at 29 °C. After incubation, the solid agar plates were inspected for the presence or absolute absence of macroscopic colony-forming units (CFU). The Minimum Bactericidal Concentration (MBC) was determined as the lowest concentration of the formulations with no visible colony growth (absolute absence). To determine the necessary concentration for 90% of growth inhibition (IC90), the resazurin dye was added to each well to a final concentration of 0.015 mg.L -1, followed by a 1 h incubation at 29 °C (here, blue-colored resazurin is reduced to resorufin, a fluorescent, pink-colored compound, when in the presence of active respiring bacterial cells). Blank wells (formulation without bacterial inoculum) were used to ensure the formulation would not reduce the dye. The quantification of resorufin was performed in a microtitre plate reader (Synergy H1, BioTek, Winooski, USA) set to excitation and wavelengths of 530 and 590 nm, respectively. All experiments were performed in triplicates in three distinct events. The formulation with lowest MBC/IC90 for each surrogate was deemed the most promising and therefore, was selected for the next steps.

The maintenance of antibacterial activity by the selected formulation was evaluated over a period of 120 days. Aliquots of 15 mL were protected from UV exposure and stored under different conditions: room temperature (~25 °C), refrigerated (4 °C), and inside the greenhouse (average temperature of 30 °C). After 30, 60, 90 and 120 days, the formula pH and antibacterial activity against surrogates were evaluated as described above.

Physico-chemical and morphological characterization

Dynamic light scattering and multi-angle particle sizing

Dynamic light scattering (DLS), Multi-angle particle sizing (MAPS), and electrophoretic light scattering (ELS) measurements were performed in a Litesizer DLS 701 instrument (Anton Paar GmbH, Graz, Austria), with Kalliope software (Ver. 4.6.0). The hydrodynamic diameter and PDI were determined using the following angles: forward Scattering (15°), side Scattering (90°) and back scattering (175°). Samples were diluted 1:50 (v/v) in ultrapure water, in triplicates, and measurements were taken at 25°C. Zeta potential values were obtained from the electrophoretic mobility data measurements, using Smoluchowski approximation.

Furthermore, the colloidal nature of selected formulation was qualitatively assessed through the Tyndall Effect. The assay was performed by exposing the sample in transparent glass containers to a directional red laser beam. The visual assessment was conducted using three distinct sample conditions: deionized water as a negative control, the formulation at a high concentration of 10% (v/v), and the formulation diluted to 1% (v/v). The 1% (v/v) dilution was specifically prepared to allow the observation of a sharp, continuous, and well-defined light path across the liquid, which serves as a visual confirmation of the nanometric dispersion.

Scanning electron microscopy

The morphological characteristics of the selected formulation were visualized using Scanning Electron Microscopy (SEM). A sample aliquot was diluted (40x and 100x), and a drop was added to steel stub covered with carbon tape. Then, the samples were left to dry in a vacuum sealed desiccator. Stubs were then sputter-coated with a 5.3 nm thick layer of gold (Elabasy et al., 2019).

Micrographs were obtained in a Zeiss Microscope EVO Ma15 (Zeiss) equipped with an EDS Bruker Xflash 6/10 (Bruker), operating at an accelerating voltage of 25 kV and working distance (WD) of 8.5 mm. Observations were performed using a secondary electron (SE) detector to obtain high-resolution topographical contrast of the particles. Analysis was performed at the Scanning Electron Laboratory (Laboratório de Microscopia Eletrônica de Varredura e Catodoluminescência (LMEV – UNESP, Rio Claro, Brazil)).

Rheological properties

Rheological measurements were performed using an Anton Paar MCR 92 rheometer (Anton Paar GmbH, Graz, Austria) equipped with a CC39 concentric cylinder geometry and a C-CC39/T200/XL cup (cup diameter: 42 mm). Temperature was maintained at 25 ± 0.1 °C using the instrument’s temperature control system, except during the temperature ramp assay. Data acquisition was executed via RheoCompass™ software (version 1.32.0.0, Anton Paar).

To characterize the flow behaviour of the formulation, steady shear viscosity curves were obtained by applying a logarithmic shear rate sweep utilizing steady-state sensing. The viscosity curves were analysed using the power-law mathematical model, where the value of n is used to define the nature of fluids (n < 1: shear-thinning nature; n = 1: Newtonian fluid; n > 1: shear-thickening nature; Asensio et al., 2020). Additionally, a temperature ramp assay was carried out at a constant shear rate to evaluate the thermal stability and the temperature-dependent viscosity profile of the system, simulating stability through storage conditions (Heating phase: 20 to 50 °C; Cooling phase: 50 to 20 °C).

Plant infection and application of the formulation

Citrus seedlings (Citrus sinensis (L.) Osbeck, Valencia variety, grafted in Swingle rootstocks) with approximately 60 cm height, commercially acquired from the same lot (Citrograf Mudas, Araras, SP, Brazil), were pruned to stimulate the emergence of young shoots, which are essential for vector feeding and pathogen acquisition. Seedlings were kept in a greenhouse in 7-liters plastic bags with pinus substrate (Multiplant Citrus, Terra do Paraíso, Holambra, SP, Brazil) with automated fertigation (Amazon AgroSciences Citrus 100, 200mL per day). When plant shoots reached stages V2-V3, characterized by fully expanded young shoots (Cifuentes-Arenas et al., 2018), they were transferred to a controlled infection room with monitored temperature, photoperiod, and humidity (Supplementary Figures 1, 2).

For each seedling, the branches containing the young shoots were marked prior to the infection. Five infective (C Las positive) Diaphorina citri adults, provided by Fund for Citrus Protection, FUNDECITRUS (Araraquara, São Paulo - Brazil), were placed on each shoot, and the shoot was covered by a fine-meshed fabric, to allow respiration and prevent escape. After 72 h the insects were removed, and frozen (-80 °C) to ensure their infectivity, if needed. The plants were left in the acclimatized room for 40 days, to allow bacterial establishment and ascertain that no egg deposition was performed in those branches, preventing possible contamination of other seedlings in the greenhouse. Sixty days after inoculation, leaves from marked branches were collected and analyzed for the presence of C Las as described in the DNA extraction and qPCR procedures detailed at topic 2.7. To ensure the plants were infected, a cut-off of Ct values ≤ 34 was considered positive for C Las (Alves et al., 2021).

Prior to the application of treatments, seedlings were randomly assigned to their experimental groups and allocated in the greenhouse following a completely randomized design. In each experiment, 20 plants were used, with 10 being treated with the formulation, and 10 being treated only with water, totaling 40 plants for both experiments. To evaluate the effectiveness of the developed formulation as a pre-symptomatic intervention, a shock-treatment schedule was implemented (Figure 1). The formulation was diluted to its MBC value and approximately 150 mL was added to each seedling substrate. Plants were subjected to an Initial Phase consisting of three drench applications within the first 7 days (days 0, 3, and 6) to mitigate early bacterial infection. This was followed by the Mid Phase, with two applications at 7-days intervals (days 14 and 21) and a Maintenance Phase of two applications at 14-days intervals (days 35 and 42). Leaf sampling for qPCR analysis was conducted on day 60 (Week 9) to assess the long-term reduction of C Las titre after treatment. The first experiment began in March 2025, and the second at end of June 2025.

Assessment of C Las bacterial titre by qPCR

Leaf samples were collected from previously marked branches of each plant at defined time points following inoculation. Total DNA was extracted according to Murray and Thompson (1980), quantified, and adjusted to a concentration between 100 and 300 ng µL - ¹ prior to analysis. Quantification of Candidatus Liberibacter asiaticus (C Las) was performed by quantitative PCR (qPCR) following the protocol described by Li et al. (2006), which is the diagnostic method formally recommended as the preferred screening test by the (EPPO, 2021). Reactions were carried out in a final volume of 12 µL containing 6 µL of NZYSupreme qPCR Probe Master Mix (NZYtech, Lisbon, Portugal), along with specific primers and probe targeting C Las (Supplementary Table 1). Amplifications were performed using a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems). Absolute quantification was performed using a standard curve generated from serial dilutions of known concentrations of C Las DNA, obtained through a constructed plasmid with target sequence (Supplementary Figure 3). Bacterial titres were expressed as log 10 of target gene copies per ng of total DNA.

Ecotoxicological assessment

Tenebrio molitor

Acute toxicity was evaluated using Tenebrio molitor (Linnaeus) larvae (Coleoptera: Tenebrionidae), an emerging model organism in agricultural toxicology due to its cost-effectiveness and reliability. Larvae were obtained from a local commercial supplier and acclimated under laboratory conditions for 24 h prior to the bioassays. During acclimation, larvae were maintained in plastic rearing containers containing wheat bran as the primary nutritional substrate, with fresh carrot slices provided as food and a source of moisture. Containers were kept at 25–26 °C under a 12 h light/12 h dark photoperiod. Prior to the bioassays, larvae were standardized according to size and weight. A volume of 1 µL of each formulation, previously diluted in acetone, was topically applied to the dorsal thorax cuticle using a micropipette, as described by Plata-Rueda et al. (2017). Two concentrations were tested: the minimum bactericidal concentration (MBC) determined in vitro and a tenfold higher concentration. A commercial insecticide (Pirate ®, BASF, São Paulo, Brazil) was used at 1 µL·mL - ¹ as the positive control, corresponding to the manufacturer’s recommended field dose. The negative control consisted of acetone only. After treatment, larvae were individually maintained in sterile Petri dishes without food, at 25 °C, 65% RH (%), and mortality was assessed after a 72-hour period. Mortality was defined as the absence of movement after mechanical stimulation, indicating lethal effects from direct exposure to the formulations. Three experimental replicates were performed for each treatment, consisting of 10 individuals per replicate.

Melipona scutellaris

Stingless bees of the species Melipona scutellaris Latreille were collected from three colonies maintained in a meliponary at São Paulo State University (UNESP), Bioscience Institute, Rio Claro campus (22°23′49″ S, 47°32′37″ W). Bees were placed in 250 mL plastic cages with lateral perforations to allow air exchange, with 10 bees per cage, and maintained in a biochemical oxygen demand (B.O.D.) incubator at 28 ± 2 °C, 70 ± 5% relative humidity, under constant darkness for 24 h for acclimatization. Microtubes with lateral perforations containing sucrose solution (50% w/v) were provided as a food source.

The experimental design consisted of four groups: an untreated control group (30 bees; 10 bees from each colony), an acetone vehicle control group (30 bees; 10 bees from each colony), and two V13 exposed groups treated with 0.5% (v/v), corresponding to the proposed dose used in the in planta assays, and 5% (v/v), corresponding to ten times the proposed dose (30 bees per treatment; 10 bees from each colony)., corresponding to the field application dose. Bees were randomly allocated to the experimental groups while maintaining equal representation from each colony. The formulations were diluted in acetone for topical exposure, and each bee received 1 µL of the corresponding treatment solution applied to the thorax using a Hamilton microsyringe (50 µL; Hamilton Company, Reno, NV, USA) coupled to a dispenser.

Prior to treatment, forager bees were cold-anesthetized at -20 °C for 70 s. The untreated control group was subjected only to cold anesthesia, whereas the vehicle-control group received acetone without the test formulation. Mortality was recorded daily, and the sucrose solution was replaced as needed to ensure continuous food availability. The assay was conducted until the death of all individuals, after which the data were subjected to statistical analysis.

Data analysis and statistics

The antibacterial activity for 90% growth inhibition (IC90) was determined through a non-linear regression curve of variable slope:

where Y = 90.

MBC data were assessed visually and determined as the most predominant concentration observed. All antibacterial data were obtained from three independent experiments consistent of three replicates each. A confidence interval of 95% (CI95%) was considered.

Rheological data were obtained using RheoCompass™ software (v.1.30, Anton Paar). Statistical analysis and curve fitting were performed using GraphPad Prism software v. 6.0 (GraphPad Software, San Diego, CA, USA). For the flow curve, linear regression was used to determine the viscosity and coefficient of determination. For the temperature ramp, data were smoothed using 8-node spline interpolation. The Viscosity Recovery Index (VRI) was calculated as the percentage ratio between the final and initial viscosity at 20 °C.

The effectiveness of drenching treatment was assessed in two distinct events. In both cases, data were assessed using a Linear Mixed-Effect Model including Time, Treatment and Time x Treatment interaction as factors. Differences between untreated and treated groups in specific time-points were verified using Sidak’s multiple comparison test. A p -value of <0.05 was considered.

Ecotoxicological data regarding T. molitor tests were performed using binary categorical values (dead/alive) and organized as contingency tables. Differences in mortality were assessed using the Chi-square test, with pairwise comparisons investigated through Fisher’s test. Survival data regarding M. scutellaris was analyzed using the Kaplan-Meier method to estimate survival curves for each treatment. Differences among groups were evaluated using the log-rank test. Median survival time (TL 50) was calculated for each group. Additionally, a Cox proportional hazards model was fitted to estimate hazard ratios among treatments. All analyses were performed using R software (R Core Team), with the survival and survminer packages. Statistical significance was set at p < 0.05.

Antimicrobial activity of formulations

After the initial formulation round, nine prototypes maintained their stability within the first 24 h period (no phase separation). These formulations were produced using hexyl gallate, eugenol, and O. vulgare essential oil as active compounds.

All formulations were screened using REMA and MBC assays against R. tropici and R. phaseoli (Table 1). The REMA and MBC assays measure the growth inhibition potential and the bactericidal dose, respectively.

The results revealed variable antimicrobial activities depending on the formulation composition and the bacterial species evaluated. Considering the strain R. tropici, formulations V11 and V13 showed the highest growth inhibition efficacy, in which V13 displayed the lowest IC90 (0.14%, CI95%: 0.17~0.12) and MBC (0.25%) values when compared with V11 (IC90 and MBC values of 0.46%, CI95%: 0.66~0.32, and 0.5%, respectively). The remaining formulations showed MBC values above 2%.

When we tested R. phaseoli, the results observed were more heterogeneous. Some formulations, including V3, V4, and V11, exhibited strong bactericidal activities (MBC <0.25%). In contrast, V12 showed the lowest IC90 (0.02%) but with no corresponding bactericidal effect (MBC >2%), which suggests a predominantly bacteriostatic action. V13 also demonstrated consistent antimicrobial activity against R. phaseoli (IC90 = 0.28%, CI95%: 0.39~0.2; MBC = 0.5%), reinforcing its broader-spectrum potential. Overall, V13 was identified as the most effective formulation, combining low inhibitory and bactericidal concentrations with consistent performance against both species. These findings supported the selection of V13 for further characterization, stability, toxicity, and in planta assays.

The maintenance of the antibacterial activity of V13 was assessed over a period of 120 days using different storage conditions, and by monitoring its bactericidal activity against R. tropici and R. phaseoli (Supplementary Table 2). The formulation kept consistent antimicrobial activity throughout the evaluated period, with only minor variations in MBC values considering the different storage conditions and time points tested. For both bacterial species, MBC values remained within a narrow range (0.5-1%), indicating no evident loss of bactericidal action. Additionally, during the 120 days of evaluation, pH values indicated the maintenance of a slightly acidic formula, with all storage conditions measurements fluctuating around the pH of 5 (Supplementary Figure 4).

Physicochemical and morphological characterization

Dynamic light scattering (DLS) analysis indicated the presence of a multimodal size population. The intensity-weighted analysis revealed the existence of a primary population with hydrodynamic diameters around 50 nm, with secondary peaks ranging from 280 to 440 nm, and a tertiary micrometric population with an average size of around 2.1 µm (Figure 2A). Conversely, the number-weighted distribution exhibited a highly homogenous, monomodal profile around 50 nm, which accounted for about 98.5% of particles, with only 0.02% of particles present at the micrometric scale. The system maintained a polydispersity index (PDI) of ≤ 0.31, indicating a moderately polydisperse system with a relatively broad size distribution. Furthermore, Zeta potential measurements recorded a mean value of -17.5 ± 0.43 mV (Supplementary Tables 3, 4).

Scanning electron microscopy captured the presence of micrometric and nanometric particles. The micrographs revealed micrometric particles and/or aggregates (around 10 µm diameters, as seen in Figure 2B), and the predominance of small particles dispersed (from 40 to 200 nm, approximately), further corroborating the majority of nanometric population. Individual micrographs of the nanometric particles were attempted, as seen in Supplementary Figure 5. Additionally, the colloidal nature of the formulation was qualitatively assessed by the Tyndall effect using a directional laser through the formulation at various dilutions. Figure 2C reveals a clear, directional and well-defined path across the sample, again corroborating the colloidal nanometric dispersion. While strictly qualitative, this visual assessment complements metrics provided by DLS and rheology. It is important to note that SEM sample preparation involves a dehydration step, which can promote particle coalescence and aggregate formation that do not fully reflect the formulation’s native state in aqueous suspension. Accordingly, SEM data should be interpreted as a complementary morphological observation rather than a standalone measure of particle size distribution, in conjunction with DLS data, which was obtained without a drying step.

Rheological data

Analysis performed on rotational rheometer revealed that formulation V13 system can be characterized as Newtonian fluid (Figure 3A). The linear relation between Shear Stress and Shear Rate (R 2 = 0.9591) along with a flow behavior index (n) of 1.113 (derived from the flow curve), confirmed the near Newtonian characteristics of the system and the absence of yield stress. Accordingly, the steady shear profile (Figure 3B) displayed a constant plateau of low viscosity (approximately 1.4 mPa.s at 20 °C) within the laminar flow regime, also corroborating the Newtonian model.

The temperature sweep performed (20 to 50 °C) (Figure 3C) revealed the thermal stability of the V13 formulation. The slight decrease in viscosity measurements observed during the heating cycle, followed by the recovery during the cooling phase, up to 83% viscosity recovery, and overlapping of curves, indicated no thermal hysteresis. These data suggest that V13 is stable against heat-induced coalescence and would exhibit optimal flow for agricultural applications.

C Las population monitored over time

The effect of V13 on the bacterial titre of C Las-infected Citrus sinensis seedlings was monitored over time by using qPCR (Figure 4).

In the first experiment (Figure 4A; Supplementary Figure 6A), the bacterial titre in untreated plants displayed a general increasing trend over time, whereas V13-treated plants maintained consistently lower titre across the same evaluated time points. Notably, at later stages of the experiment (240 and 300 days), treated plants exhibited reduced bacterial loads compared to the untreated group, with statistically significant differences observed at 300 days. Analysis showed that the interaction between time and treatment was highly significant (p = 0.0015), as well as the time variable (p = 0.0043), while the sole treatment variable was non-significant (p = 0.1282). These results highlight the progression of the disease, with increasing bacterial titre overtime, and reveal the effect of V13 application, statistically reducing up to 1.29 log 10 bacterial load with continuous applications. This overall reduction reflects the fact that 70% of the treated plants reached bacterial titers at or near the limit of detection (LOD) after 300 days of continuous applications.

These findings were validated in the second experiment (Figure 4B; Supplementary Figure 6B), which was initiated under cooler winter conditions, characterized by milder temperature and shorter photoperiods. While untreated plants showed a progressive increase in C Las titre, V13-treated plants maintained substantially lower bacterial levels throughout the experiment. In this instance, not only the interaction between time and V13 application was considered significant (p < 0.0001), but the treatment effect alone was also deemed significant (p = 0.0039). This variation in the standalone effect of the treatment could be attributed to seasonal environmental variations between the trials, as alterations in the respiration rates, and photoassimilate flow can influence the bacterial colonization. In the second experiment, the V13 application resulted in a reduction of 2.46 log 10 in the bacterial load relative to the untreated plants after 300 days of experiments. This overall reduction was driven by the fact that 90% of the treated plants reached bacterial titers at or near the limit of detection (LOD) at the endpoint. Significant differences between treated and untreated groups were detected at 180, 240 and 300 days, reinforcing the effect of the treatment in limiting bacterial proliferation, especially in the early stages of infection, with lower bacterial titers. Still, analysis of individual plants revealed a heterogenous response in bacteria titers to V13 treatment over the 300-day trial. Rather than a uniform shift across the group, the treated population separated into a highly responsive subset where bacterial titers dropped to or near the limit of detection (n = 7 in trial A and n = 9 in trial B). At the 300-day endpoint 70% of treated plants in trial A and 90% of treated plants in trial B achieved bacterial titers at or near the LOD, with this responsive subset showing a median log 10 reduction of 3.44 relative to matched controls, indicating a titre-reducing effect of V13 on C Las.”

Ecotoxicological profile against indicator species

Considering the proposed application of the V13 formulation via root drenching, a multi-compartment ecotoxicological approach was developed. The concentration proposed for field application was based on the minimum bactericidal concentration (MBC) obtained in vitro (0.5%, v/v). To evaluate the worst-case exposure scenario, T. molitor larvae were also exposed to a tenfold higher concentration (5.0%, v/v). No acute toxicological effects were observed at either concentration (Figure 5A). The results demonstrated that the negative control showed low average mortality (3.3%). In contrast, positive control resulted in significantly higher mortality (60%), validating the sensitivity and effectiveness of the method used. Larvae treated with V13 formulation at a concentration of 0.5% showed no mortality after 72 hours, while at 5% mortality was less than 1%, indicating no evidence of acute toxicity under the assay conditions.

For M. scutellaris, a native stingless bee, known to use clay/soil in hive construction, survival analysis showed no significant differences among treatments (log-rank test, p = 0.6). Median survival time (TL 50) was 19 days for the control, 19 days for the acetone control, 18.5 days for the 0.5% treatment, and 16.5 days for the 5% treatment (Figure 5B). The Cox proportional hazards model also showed no significant differences among treatments (p = 0.6). Overall, V13 exposure did not significantly affect the survival of M. scutellaris under the tested conditions.

Management of HLB is one of the greatest challenges in current citriculture. Due to the phloem-restricted nature of C Las, the delivery of antibacterial compounds to infected tissues is seriously compromised (Bové, 2006; Ghosh et al., 2022). In recent years, colloidal and nanostructured formulations have been investigated as possible strategies for targeted transport of active molecules to plant vascular tissues (Lowry et al., 2024; Sangwan and Singh, 2025). These nano delivery systems combine the protection of active molecules, which are highly sensitive to environmental degradation, with enhanced penetration capabilities, reaching microbial targets within plant tissue (Sangwan and Singh, 2025). For instance, Yang et al. (2015) demonstrated that nanoemulsions loaded with ampicillin were able to significantly reduce C Las titre when applied directly to the leaves, outperforming the unformulated antibiotic.

Regarding the particle size of V13 formulation, with a population ranging from 280 to 440 nm, the presence of multiple particle size populations is hypothesized to represent a functional asset rather than a physical artifact, although direct evidence of particle translocation within plant tissue was not evaluated in the present study. In plant biology, particle dimensions heavily dictate how a formulation navigates complex barriers, such as the waxy leaf cuticle and stomatal pores to cell wall junctions and apoplastic pathways (Xu et al., 2026). When within plant tissue, these particles can move by diffusion, bulk flow and phloem loading, as described for nanoscale carriers in other plant systems (Wang et al., 2023). While the nanoscale fraction is ideal for diffusing through microscopic structural gaps in plants, and percolating more efficiently through soil pores, the larger particles and aggregates are likely to play a complementary role. Particles in the micrometric scale can improve physical retention within the soil matrix, enhance adhesion to the tissue surface, and prevent soil leaching, possibly providing a reservoir of active molecules in the soil (Xu et al., 2026; Su et al., 2023; Kah et al., 2013). While number-weighted DLS data indicate that the micrometric population represents a small fraction of total particle count (~ 0.02%), the cubic relationship between particle radius and volume implies this population could constitute a disproportionately larger fraction of total formulated mass, which was not directly quantified in this study. This is consistent with reports that micron-scale carriers (e.g., ~20 µm lignin microparticles) can increase dosing time and reduce soil leaching of agrochemicals such as atrazine (Taverna et al., 2018), supporting the hypothesis that the micrometric fraction of V13 may function similarly as a localized reservoir, though this remains to be experimentally confirmed for the present formulation.

Concerning physical stability from V13 formulation, it is notable that despite displaying an absolute Zeta potential value usually associated with low electrostatic stability (-17.5 mV ± 0.43 mV), V13 is a complex formulation containing. Thus, V13 is a complex formulation containing soy lecithin and polysorbate, which provide interfacial and steric stabilization alongside electrostatic contributions. Its colloidal stability should therefore not be interpreted through the conventional |ζ| ≥ 30 mV criterion, which applies specifically to dispersions stabilized predominantly by electrostatic repulsion. In accordance with this, the negative potential measured despite the presence of cationic polymers indicates a lecithin/gallate-dominated interface. The V13 formulation maintained full bactericidal activity over 120 days and showed no thermal instability in the temperature-ramp rheology, direct evidence that the low absolute zeta potential did not compromise stability (Moreno-Trejo et al., 2019).

Furthermore, viscosity is a key parameter in agricultural formulations because it influences not only product stability, but also its behavior during field applications. Adequate rheological properties, such as stable viscosity in shear stress conditions, indicate a high-integrity formulation (Mustafa and Hussein, 2020). The viscosity data of V13 indicating the absence of pronounced thermal hysteresis not only indicates a stable suspension but also favors the application of the product in agronomical setups. Beyond facilitating soil navigation, nanometric-sized populations could play a crucial role by ensuring colloidal stability and bioavailability of hydrophobic particles (e.g. essential oils) in the aqueous phase (Hunde et al., 2023). Regarding compatibility with sprayers, our rheological data indicate that the V13 formulation behaves as a nearly Newtonian fluid (flow behavior index n = 1.113), exhibiting no yield stress and maintaining low, stable viscosity (~1.4 mPa·s at 20 °C) across the tested shear range. This flow profile is compatible with agricultural equipment. Finally, we highlight that micrometer-scale formulations have been described as potential localized reservoirs capable of prolonging release times and reducing soil leaching of agrochemicals such as atrazine (Taverna et al., 2018), which could offer a functional advantage in soil applications.

Essential oil-derived molecules, such as eugenol and carvacrol, used in the proposed formula, have been classified as transdermal facilitating agents for plant tissue permeability. These lipophilic molecules can disrupt plant cell membranes by intercalating with the densely packed lipids in the membrane, significantly increasing cellular permeability (Maffei et al., 2001). In the HLB management context, reports indicate that geraniol was able to enhance fludioxonil and tetracycline penetration in citrus leaves up to 15 and 20-fold, respectively (Chen et al., 2025).

Some authors described techniques such as “bark painting” or trunk injection as more efficient than root drenching, often used to deliver oxytetracycline directly into the plant vascular system, suggesting that the root absorption can offer challenges to particle permeation and compound transport (Yang et al., 2016; Killiny et al., 2020; Albrecht et al., 2025). Yet, others indicated that even in the presence of adjuvants, broad-spectrum antibiotics such as oxytetracycline, and streptomycin, have low permeability through leaf cuticles, resulting in no significant changes in C Las titre (Vincent et al., 2022). This suggests that while some techniques can offer specific advantages, there is no singular solution for accessing plant tissues. Efficacy ultimately depends on pairing formula composition with the optimal route of application. In this context, our formulation strategy incorporates surfactants and transdermal natural molecules to facilitate the permeation of hydrophobic essential oils in plant tissue. Additionally, the multimodal size distribution can promote adequate soil distribution and release of active molecules. Together, these characteristics suggest that V13 offers a promising approach to overcome the challenges of root absorption (Maffei et al., 2001; Moreno-Trejo et al., 2019).

Consistent with the enhanced permeability and composition of our colloidal system, in planta assays demonstrated the practical effect of the V13 formulation. Over the 300-day period, the drench application resulted in a reduction of up to 2.46 log 10 in the bacterial load of infected plants, when compared to untreated controls, with 90% of treated plants reaching bacterial titers at or near the LOD. This reduction is considered significant, as the higher bacterial load is not only related to the development of the disease but also influences the efficiency of pathogen transmission by the psyllid vector (Lopes and Cifuentes-Arenas, 2021). Beyond direct antibacterial activity, the essential oil and essential oil-derived components of V13 may also trigger systemic plant defense mechanisms. Despite the disruption of the plant cell membrane by these lipophilic molecules being usually temporary, it can lead to membrane depolarization and Ca 2+ leakage. This calcium influx could potentially activate downstream signaling cascades, inducing oxidative bursts, with the generation of reactive oxygen species (ROS) (Maffei et al., 2001). Hypothetically, this process could elicit endogenous salicylic and jasmonic acid pathways, priming the immune response of the plants. A recent work in which oregano essential oil, present in V13 formulation, was used in grapevines indicated that its ability to trigger the plant immune system enhanced its overall antimicrobial effect (Rienth et al., 2019).

Regarding the safety of the repeated drench-application schedule, no visible signs of phytotoxicity (e.g., leaf necrosis, wilting, or growth arrest) were observed in treated seedlings throughout the shock-treatment phase (days 0, 3, and 6) or the subsequent 300-day monitoring period, and all treated plants survived to the end of the evaluation. However, quantitative phytotoxicity endpoints, such as root biomass, shoot growth, and chlorophyll content, were not assessed in the present study, as this proof-of-concept work focused primarily on antimicrobial efficacy. A dedicated assessment is identified as a priority for subsequent stages of formulation development.

In the search for sustainable alternatives to traditional broad-spectrum antibiotics (e.g. oxytetracycline and streptomycin), current research has increasingly focused on molecules from biological sources as control strategies. Huang et al. (2021) demonstrated that stable antimicrobial peptides (SAMPs) could reduce C Las titre and diminish symptoms in infected plants. Similarly, the use of lipopeptides and phytohormones from Bacillus amyloliquefaciens rendered reductions of approximately 52% in bacterial load after successive applications (Ding et al., 2024). Alongside microorganism-derived molecules, botanical active ingredients such as essential oils have, as well as semi-synthetic derivatives like hexyl gallate, been extensively investigated as control agents for phytopathogens (Cavalca et al., 2020; Braun et al., 2023; Marin et al., 2024a; Llanos et al., 2026). However, direct field application of these molecules is hindered by their high volatility, low physical retention, and potential for undesired allelopathic or phytotoxic effects (Marin et al., 2024b). Therefore, the innovative potential of V13 does not rely on the discovery of new active molecules, but on the manufacturing of an appropriate colloidal system, tailored to overcome these physicochemical limitations to reduce C Las development in infected citrus.

While reducing C Las population development inside the plant is crucial, sustainable HLB management also relies on the control of the psyllid vector and the development of more effective agrochemicals for this goal (Cui et al., 2025). While this field of vector control research is no stranger to ecotoxicological analysis (Chen et al., 2017), most studies about antibacterial agents for in planta application are only focused on citrus tree toxicity, the rhizosphere microbiome impact, or non-target eukaryotic genes (Castellano-Hinojosa et al., 2024). The ecotoxicological data obtained under the tested conditions revealed no acute toxicity in T. molitor larvae and no significant effects on the survival of M. scutellaris foragers. These findings provide an initial safety characterization of V13 towards the two non-target insect species evaluated.

Nevertheless, these results should be interpreted within the scope of the assays performed. The present assessment focused on mortality in T. molitor over 72 h and survival in M. scutellaris following topical exposure, and therefore does not encompass potential sublethal effects, alternative exposure routes, or effects on other non-target organisms and environmental compartments. In particular, exposure of pollinators through contaminated nectar or pollen would depend on the uptake and translocation of the formulation or its active compounds within treated plants, which were not investigated in the present study. Similarly, potential effects on soil microbial communities and other components of the rhizosphere were beyond the scope of this initial formulation assessment. Further studies addressing these exposure pathways and endpoints will be important to support a more comprehensive environmental risk assessment of V13.

It is important to note that our formulation is not composed of traditional antibiotics, such as oxytetracycline, streptomycin or β-lactams. Instead, the use of natural and semi-synthetic compounds as active ingredients could present an advantage. While the established colloidal system works on delivering the active molecules to the bacterial target, the use of multiple active ingredients is crucial to mitigate the emergence of bacterial resistance. Essential oils, such as oregano essential oil can disrupt the bacterial membrane, leading to permeabilization and leakage (Zamuner et al., 2023). Concurrently, semi-synthetic gallate can both disrupt the membrane and affect bacterial cell division (Kopacz et al., 2019; Cavalca et al., 2020).

This study was designed as a proof-of-concept to establish the physicochemical stability and antibacterial efficacy of V13 under controlled greenhouse conditions. Consequently, several aspects relevant to commercial translation like field validation under variable environmental conditions, formulation scale-up production and economic feasibility, and the V13 fate in complex field soils, were beyond the scope of the present work. These represent necessary next steps before V13 can be considered for practical adoption in citrus orchards and are the focus of ongoing and planned studies by our group.

In conclusion, V13 formulation represents a robust, non-synthetic alternative for the agronomic management of HLB. By optimizing the interfacial properties and particle size, the formulation can interact successfully with root tissue via drench application, without the necessity of invasive and costly techniques such as trunk injections. These findings indicate that V13 can effectively reduce CLas titre following root-drenching application, while showing no evidence of acute toxicity in T. molitor and no significant effects on the survival of M. scutellaris under the tested conditions. Nevertheless, a heterogenous response in bacteria titers to V13 treatment over the 300-day trial was observed. Such variability in bacterial titres is a recognized challenge in systemic treatments in long-term greenhouse trials. Differences in individual root architecture, localized vascular colonization density of C Las prior to treatment, and micro-variations in absorption efficiency likely dictate whether systemic distribution reaches the threshold required for the complete eradication of the pathogen. These results highlight V13’s intrinsic antimicrobial capacity while emphasizing that future formulation improvements must prioritize enhanced uptake consistency. As research on this formulation progresses, further studies addressing sublethal effects, alternative exposure pathways, and other environmental compartments can provide additional information and contribute to a more comprehensive characterization of its environmental safety profile.