The virulent R. solanacearum was obtained from the Molecular Biology and Biotechnology Laboratory, Institute for Basic Sciences Technology and Innovation, Pan African University located in Juja (Kenya) [11]. Thymol, eugenol, Pentasodium tripolyphosphate (TPP), chitosan with low molecular weight (75% - 85% degree of deacetylation) used as a coating material were purchased from Sigma-Aldrich, Hamburg, Germany. Tween 80, acetic acid, hydrochloric acid and ethanol (95%) were purchased from Legacy Lab Africa.

Encapsulation of eugenol and thymol in chitosan with TPP was performed using the two step oil-in-water emulsion and ionic gelation method [27]. Tween 80 was used as a surfactant and chitosan solution (1% w/v) was prepared by shaking chitosan at room temperature overnight in 1% v/v aqueous acetic acid solution. Tween 80 (336 µl) was added to 40 ml of chitosan solution and the mixture stirred at 45˚C for 2 hours forming an emulsion. Gradually, 0.5 g of thymol was added into the stirred mixture, and agitation carried out for 20 min. Then, 40 ml of 0.5% (w/v) TPP solution was dropped slowly into the emulsion while agitating and stirring continuously at room temperature for 30 min. Collection of the formed particles was achieved through centrifuging the formed solution at 4000 rpm for 30 min at 25˚C, and then cleaned by washing three times with water. The obtained wet particles were dispersed in 25 ml of distilled water via probe sonicator for 4 min (2 s sonication and 1 s rest) in an ice bath to get a homogeneous suspension. Finally, the formed particles were freeze dried at −65˚C for 72 h. The dried nanoparticles (NPs) were then stored at 4˚C until further analysis.

The morphology and the size of the nano-capsules were analyzed by scanning electron microscope (0860000 Advance Laboratory Solutions, South Africa); sample preparation followed the method described by Hosseini et al., 2013 and Fourier-transform Infrared Spectroscopy analysis (FTIR) was used to study the chemical characteristics of the nanoparticles to determine the functional groups involved in the reaction [25]. FTIR analyses for pure chitosan, thymol, eugenol together with CNPs (loaded or unloaded with thymol and eugenol) were recorded from electromagnetic spectrum range of 500 - 4000 cm⁻¹ by Shimadzu FTIR-8400 (Kyoto, Japan). The samples were prepared by grinding the dried nanoparticles with potassium bromide before pressing them to form disks [28]. For each spectrum, 16 scans at a resolution of 4 cm⁻¹ were obtained.

The amount of thymol and eugenol encapsulated in chitosan nanoparticles (CNPs) was determined by UV-vis spectrophotometry technique as described previously [25]. The sample was prepared by mixing 10 mg of thymol-loaded chitosan nanoparticles (TCNPs) in 4 ml of 2 M HCl then boiled at 95˚C for 30 minutes. After cooling, 2 ml of ethanol was then added to the homogenous mixture before being centrifuged at 9000 rpm for 5 min at 25˚C. The procedure was repeated for eugenol-loaded-chitosan nanoparticles (ECNPs). The supernatant was then analyzed for the thymol and eugenol content using UV-vis spectrophotometry at the wavelengths of 292.5 nm and 295 nm respectively. The amount of thymol and eugenol was calculated by calibration curves of free thymol and eugenol; CNPs was similarly treated and used as a blank and standard calibration curve of both eugenol and thymol were established using five standard solutions with 0.02%, 0.04%, 0.06%, 0.08%, and 0.10% w/v diluted in absolute ethanol. Each batch samples were measured in triplicate and the loading capacity (LC) along with encapsulation efficiency (EE) of the compounds were estimated from Equations (1) and (2) respectively [24].

EE ( % ) = Weight of loaded compound Weight of initial compound × 100 (1)

LC ( % ) = Weight of the loaded compound Weight of nanoparticles after freeze drying × 100 (2)

The release characteristics of thymol and eugenol from CNPs was assessed in phosphate buffer saline (PBS) at pH 1.5, 5.5 and 7.4 as described by Shetta et al., 2019 [29]. Freeze dried nanoparticles (40 mg) were put in a dialysis bag (12,000 - 14,000 KDa) containing 2 ml of PBS and was then incubated in a 50 ml tube having 15 ml of the same release media at room temperature under gentle shaking. At every 12-hour interval, 5 ml of release media was taken for analysis, with an addition of an equal volume of fresh release media. The total cumulative amount of thymol and eugenol (mg) in the volume of the release medium (ml) that were encapsulated in NPs, was quantified spectrophotometrically. The release was quantified as follows:

Release (%) = [Released oil/Total oil] × 100 [30]

The efficacy of encapsulated eugenol and thymol in CNPs against R. solanacearum was investigated using broth dilution and colony counting methods as described by Amro, 2017 [29]. The EOs and nano-encapsulates were first sterilized by keeping them under UV light for 2 hours while R. solanacearum was cultured on Casamino acid-Peptone-Glucose (CPG) broth aerobically for 24 hours at 28˚C. After overnight growth, 2.5 ml of R. solanacearum culture containing 5% DMSO was taken into falcon tubes containing 5 mg of CNPs, TCNPs, ECNPs and corresponding amounts of pure eugenol and thymol for an incubation at 28˚C in a shaker at 250 rpm for 12 hours. The positive control consisted of CPG broth with R. solanacearum in order to check the growth of the bacteria while the negative control was CPG broth alone in order to check for the sterility of the media used in the experiment. Serial dilution was then prepared from the falcon tubes by addition of CPG broth and then 50 µL of the 7^th fold dilution was spread on the CPG agar and cultured at 28˚C for 24 hours and the colonies quantified with the help of a colony counter. The antibacterial activity was estimated through the reduction in numbers of the bacterial colonies after introducing the sample compared with the positive control. All treatments were determined in triplicate and average value was used in determining the percentage inhibition.

Inhibition of R. solanacearum growth (%) = C − A C × 100

where C and A are the bacterial colonies of positive control and treated plates respectively.

The MIC of thymol, eugenol, ECNP and TCNP in relation to R. solanacearum were measured in triplicate by 96-well broth micro-dilution method using resazurin dye [11]. For eugenol and thymol, the MIC was determined based on concentration ranging from 300 to 100 μg/ml (300, 275, 250, 225, 200, 175, 150, 125 and 100) as previously determined from a pre-experiment. However, for TCNP and ECNP it was determined using a concentration range of 720 µg/ml to 2.8 µg/ml (720, 360, 180, 90, 45, 22.25, 11.125, 5.6 and 2.8) in a CPG broth medium using a 96 well plate. The concentration range was chosen based on preliminary studies. To each well, 10 μL of R. solanacearum (10⁸ CFU/ml) was then added and then it was incubated at 28˚C for 24 hours. After incubation, 20 µL of resazurin solution at 0.015 % m/v was added per well and the results recorded after further incubation at 28˚C for 1 h. The lowest concentration at which no color change occurred (blue color retained) was considered as the MIC [31]. Positive control consisted of 100 µL of CPG broth containing 10 μL of R. solanacearum while the negative control contained 100 μL of broth only [21]. Each experiment was carried out in duplicate and in three independent repeats.

The prepared CNPs, ECNPs and TCNPs were characterized using Scanning Electron Microscope, Fourier-transform Infrared Spectroscopy analysis and UV-Vis spectrophotometry. SEM images of both TCNPs and ECNPs demonstrated distinct particles with spherical shape (Figure 1). Similar results were observed when oregano EO was encapsulated in CNPs [25]. The size of polymeric nanoparticles prepared from biocompatible polymers such as chitosan had been investigated and determined to range between 10 - 1000 nm [32]. In this study, the average size of CNPs was 375 nm while those of TCNP and ECNP were 590 nm and 555 nm respectively (Figure 1). The smaller size of CNPs in comparison to the EO-loaded nanoparticles is due to the loading of thymol and eugenol in CNPs [25]. The nanoparticle size obtained were in the same range

with a similar study which reported a range of 532.5 - 716.6 nm nanocapsules size when EO major component carvacrol was encapsulated in CNPs [24]. Rasaee et al., 2016 also obtained nanocapsules size range of 135 - 729 nm when leaf extract of Ocimum basilicum was encapsulated in chitosan. Morphological studies of the particles provide valuable perspective in relation to the chemical and physical factors affecting the particle structure [21].

FTIR spectroscopic analysis was utilized to examine the chemical characteristics of chitosan, CNPs, eugenol, thymol, TCNPs and ECNPs. Chitosan showed characteristic broad peak at 3408 cm⁻¹ due to hydroxyl group and primary amine stretching. However, in CNP, this band shifted to 3423 cm⁻¹ and became wider, indicating an enhancement in hydrogen bond due to hydrostatic interaction between amino and phosphoric groups in TPP (Figure 2) [28]. The peak at 1117 cm⁻¹ indicated the amino group of chitosan but this peak shifted to 1121 cm^–1 in CNPs. The shift is attributed to interaction between the amino group and phosphate anion in TPP signifying the formation of nanoparticles [30].

Eugenol showed characteristic peaks at 3517 cm⁻¹ (OH), 2843 - 3073 cm⁻¹ (C-H stretching), 1513 cm⁻¹, 1608 cm⁻¹ and 1638 cm⁻¹ (C-C aromatic ring) (Figure 2). In comparison with the FTIR spectrum of CNP, the addition of eugenol resulted in a markedly increased intensity of the C-H stretching peak at 2931 cm⁻¹, indicating an increase in the content of ester groups, which might come from eugenol. This increment of C-H stretching peak indicate a successful encapsulation of eugenol into CNP [27].

Major peaks at 3408, 2931, and 1110 cm⁻¹ in ECNP indicated the presence of N-H, O-H and CH-OH functional groups respectively [30]. The FTIR of ECNP showed bands at 2931, 2065 and 1110 cm^–1, which were absent in CNP but present in eugenol, indicating successful incorporation of eugenol in the ECNP (Figure 3). The band attendance at 3423, 3421 and 3379 cm⁻¹ in CNP, thymol and TCNP respectively represents hydroxyl group and primary amine stretching (Figure 4). Sp3 C-H stretching appeared at 2961 cm⁻¹ in thymol and 2960 cm⁻¹ in TCNP and was absent in CNP which revealed the successful encapsulation of thymol with CNPs [33]. Amide stretching peaks appeared at 1597, 1591 and 1597 for CNP, thymol and TCNP respectively. A characteristic absorption peak at 1249 which is related to ether (C-O-C) bonds appeared only in thymol and

TCNP and was absent in CNP. Thymol and TCNP also shared a common peak at 1110 cm⁻¹ which is related to ether (C-O-C) bonds. These FTIR results confirmed the incorporation of eugenol and thymol in the chitosan-TPP system.

Calibration curves for pure thymol and eugenol were constructed and used to determine LC and EE for TCNPs and ECNPs. The generated curves for thymol and eugenol had R² of 0.996 and 0.986 respectively. The percentage amount of eugenol and thymol encapsulated in TCNP and ECNP was estimated by determining the EE and LC spectrophotometrically based on the calibration curves [24]. The EE of thymol and eugenol in TCNP and ECNP was determined as 72.9% and 71.7% while LC % was 48.3% and 49.5% respectively (Figure 5). These findings were in agreement with previous reports on the encapsulation of peppermint in CNPs [29] [34]. However, a higher encapsulation efficiency greater than 90% have also been reported when cardamom essential oil was encapsulated in chitosan nano-composites [18].

The in-vitro release study of thymol and eugenol from TCNP and ECNP was carried out for 180 hours in three different pH media (1.5, 5.5 and 7.4) to confirm the successful encapsulation of the compounds and to determine the rate at which the compounds are released from the NPs [24] (Figure 6). The release profile for both eugenol and thymol could be described as a two-step biphasic process, involving an initial burst release followed by subsequent slower release [25]. In the first step (first 12 hours), there was burst release, which could be due to diffusion of the compounds adsorbed on the surface of particle and those near the surface. In the second step however, the release was relatively slow and this

could be attributed to the diffusion of thymol and eugenol dispersed into the polymer matrix. A similar release profile had been reported previously [24]. The release of the EO components from the microcapsules was found to be pH dependent. At pH 7.4, 5.5 and 1.5, the maximum release percentages for thymol were 63.8%, 74.7% and 83.0% respectively while for eugenol, the maximum release percentages were 64.1, 78.1 and 85.6% in pH 7.4, 5.5 and 1.5 respectively. The results suggest that the rate of release of thymol and eugenol from CNPs is faster in acidic solution (pH = 1.5) than in slightly acidic (pH = 5.5) and neutral (pH = 7.4) respectively. In acidic media (pH 1.5), both thymol and eugenol were released from CNPs very quickly, and the released contents were significantly higher than what occurs at higher pH media (pH 7.4) which is most probably due to the swelling and partial dissolution of the NPs. The relatively higher release of the encapsulated compounds from the CNPs could be due to the partial dissolution and swelling of the TCNP and ECNP caused by ionic repulsion of protonated free amino groups on one chitosan chain with its neighboring chains [35]. It was also noted that the two compounds were not completely released from NPs because complete release needs complete degradations of CNPs [29].

To determine the percentage mortality of R.solanacearum in the presence of CNPs, TCNPs and ECNPs, agar dilution and colony counting methods in CPG medium was used (Figure 7) as described by Shetta et al., (2019). The percentage reduction in numbers of the bacterial colonies after the treatment with TCNPs and ECNPs when compared to the colonies of positive control (CPG media with bacteria only) were used to estimate the antibacterial activity. Positive control had the highest number of R. solanacearum colonies which indicated a normal growth of the pathogen in CPG agar medium whereas, no colonies were observed in the negative control confirming the sterility of agar medium. In the plate containing CNPs, percentage inhibition of 80% was recorded while chitosan alone showed percentage inhibition of 44%. An enhanced inhibition percentage of 92% and 94% for TCNP and ECNPs respectively when compared to the positive control was recorded (Figure 8). There was however, no

significant difference in the rate of inhibition between TCNP and ECNP. Donsì et al., 2011 also reported similar results in which the antibacterial potential of EOs were enhanced against pathogens causing food spoilage after being encapsulated with nanoparticles [36]. In another study, Kalagatur et al., 2018 reported an enhanced antifungal activity of CNPs encapsulated with Cymbopogon martinii EO on plant pathogenic fungi Fusarium graminearum [12]. The findings are further supported by a study by Khalili et al., 2015 who reported an enhanced antifungal activity of chitosan encapsulated thyme EO nanoparticles over thyme EO during safeguarding of the tomato fruit against Aspergillus flavus [37]. The enhanced antimicrobial activity of encapsulated EOs had been linked to sustained release, improved hydrophilicity, and the better penetration resulting from small size of the nanocapsules [38].

Using the minimum inhibitory concentration assay, thymol, eugenol, TCNPs and ECNPs were compared for the antimicrobial efficacy of the EO against R. solanacearum. Before encapsulation, the MIC of thymol and eugenol was determined as 175 µg/ml and 275 µg/ml respectively (Table 1). However, after encapsulation with CNPs, the MIC of thymol and eugenol against R. solanacearum reduced significantly to 22.5 µg/ml and 45 µg/ml respectively (Table 2). In a study by Van Vuuren et al., 2010, the MIC values of Melaleuca alternifolia EO ranged from 250 µg/ml against C. albicans to 2000 µg/ml against E. coli and S. aureus [39]. After encapsulation however, the MIC was lowered to values between 17 and 34 µg/ml against all the pathogens. The reduction in MIC was

Inhibited; (+): not inhibited; (−): Positive control (broth + R. solanacearum); (+ve): Negative control (broth only); (−ve).

Inhibited; (+): not inhibited; (−): Positive control (broth + R. solanacearum); (+ve): Negative control (broth only); (−ve).

attributed to the persistence of antibacterial activity due to the sustained release of compounds [40]. The reduction MIC may also be due to synergistic activity between EO compounds and CNPs [39].