The bacterial spot of stone fruits and almond is a significant disease of many Prunus species with a large and increasing distribution area. Its causative agent, Xanthomonas arboricola pv. pruni (Smith, 1903; Vauterin et al., 1995) (abbreviated as “Xap” hereafter) is included in the A2 list of pests by the European and Mediterranean Plant Protection Organization (EPPO, 2023). Xap was first identified in Hungary in 2004 (Németh, 2005). Its spread was successfully inhibited due to the applied quarantine measures, but from 2016 the pathogen was re-detected on different hosts and from several Hungarian locations (Schwarczinger et al., 2017; Schwarczinger et al., 2018; Kolozsváriné Nagy et al., 2020). Xap poses a real threat to its economically important hosts (peach, nectarine, apricot, Japanese plum, and almond) especially in areas where the climate is humid and warm during the growing season (Battilani et al., 1999). This pathogen induces water-soaked spots on the surface of leaves, fruits, and shoots that can become brown and necrotized (Civerolo and Hatting, 1993). Its significance lies in the fact that severe leaf infections lead to diminished vitality of the trees and the highly infected fruits are simply unmarketable, while twig cankers combined with early defoliation may result in complete destruction of the plant (EPPO, 2006). The management of this disease is mainly limited to prevention – i.e. the certification of propagation material, the use of less susceptible cultivars (Lamichhane, 2014) or disease forecasting models (Battilani et al., 1999; Morales et al., 2018) – and the application of copper-based pesticides (Osdaghi, 2022) and oxytetracycline (OTC) in the USA (Vidaver, 2002). However, most peach, apricot, and Japanese plum genotypes are susceptible to the bacterium (Garita-Cambronero et al., 2018) and multiple copper treatments can result in not only phytotoxicity but the development of copper tolerant or resistant plant pathogenic bacterial strains (Giovanardi et al., 2017; Cox et al., 2022). As for the other chemical pesticide, OTC resistance has been reported in Xap strains in peach orchards of the southeastern USA (Herbert et al., 2022). Therefore, the study of alternative control measures like the usage of essential oils (EOs) is of high priority.

EOs are natural complex mixtures of botanical origin carrying a distinctive scent, or essences of the originating plants, which have a history of several thousand years (EFEO/IFRA, 2015). Compared to synthetic pesticides, natural ones have beneficial features including biodegradability, target specificity, and posing no or lower risk to the environment or human health (Walia et al., 2017). The therapeutic use of biologically active volatile plant compounds includes clinical (Buckle, 2015), food preservative (Corrêa and Ferreira, 2022), and plant protection (Assadpour et al., 2023) applications. The great potential of EOs as biopesticides is based on their herbicidal (Li et al., 2020; Yasar et al., 2021), insecticidal (Isman et al., 2011; Ebadollahi et al., 2022), acaricidal (Camilo et al., 2017; Nwanade et al., 2021), nematicidal (Avato et al., 2017; Catani et al., 2023), and antimicrobial effects (Božik et al., 2017; Assadpour et al., 2023), and has been well demonstrated by usage of commercialized bioinsecticides (Isman, 2020). A variety of EOs has shown effectiveness against several Xanthomonas species (Horváth et al., 2004; Huang and Lakshman, 2010; Bajpai et al., 2011; Lucas et al., 2012; Móricz et al., 2015a; Móricz et al., 2015b; Gormez et al., 2016; Mirzaei-Najafgholi et al., 2017; Singh et al., 2017; Kumar et al., 2021; Hakalová et al., 2022; Montesinos et al., 2023) and especially against Xap (Biavati et al., 2004; Mahmoudi et al., 2010; Zhao et al., 2011; Montesinos et al., 2023).

Among in vitro antibacterial susceptibility assays, routine well and disc diffusion methods are preferably used for the examination of polar components due to the limited diffusion of lipophilic compounds to the agar (Rios et al., 1988; Móricz and Ott, 2016). Therefore, the commonly used dilution methods and bioautography are more suitable for the determination of antibacterial activity of EOs that are mixture of volatile, complex, and viscous substances generally with low solubility in water. Direct bioautography (DB), the combination of high-performance thin-layer chromatography (HPTLC) with antibacterial assays, is a sensitive, fast (it takes less than 3 h), and reproducible high-throughput screening protocol for assessing the antibacterial effects of EOs, without diffusion or solubility limits (Horváth et al., 2013; Dewanjee et al., 2015; Móricz and Ott, 2016). Furthermore, DB facilitates detection of HPTLC inhibition zones allowing estimation of the antibacterial effects of individual separated compounds that can be characterized and identified by various HPTLC hyphenations e.g. spectroscopy and mass spectrometry (Móricz and Ott, 2023).

Due to the lack of registered antibacterial compounds effective against Xap, the aim of the present study was (i) the survey of antibacterial impacts of the EOs of thyme, cinnamon, clove, rosemary, tea tree, eucalyptus, lemon grass, citronella grass, and lemon balm on Xap; and (ii) the identification of the tested EOs’s active components by the HPTLC-Xap method combined with solid-phase microextraction-gas chromatography/mass spectrometry (SPME-GC/MS).

The antibacterial activity of EOs was evaluated by MIC against the two Xap isolates using BMA in 96-well plates. Both Xap3 and XapG2 were susceptible to all investigated EOs, but to different degrees (with MIC between 31.25 and 1000 µg/mL), and generally the XapG2 isolate was similar or less sensitive to the EOs than Xap3 ( Table 1 ). The tea tree EO was the least effective providing the highest MIC value. Comparing the antibacterial potential of EOs to those of antibiotics, the strongest activity was attributed to cinnamon against both Xap3 and XapG2 with a 12.5 - and 25 -times higher MIC value than that of chloramphenicol, respectively. In the efficiency order, cinnamon was followed by thyme, clove, and lemon grass with noticeably inhibitory activity.

To identify the EO components responsible for anti-Xap activity, a DB assay was newly adapted for Xap. This method enables the detection of separated individual compounds that display Xap inhibition. The separation was carried out on HPTLC silica gel 60 F₂₅₄ layer with n-hexane – ethyl acetate (9:1, V/V), and vanillin-sulphuric acid reagent was employed for the visualization of the non-UV active EO compounds ( Figures 2A, D, G ). Generally, the bioprofiles obtained by DB using Xap3 ( Figures 2B, E, H ) or XapG2 ( Figures 2C, F, I ) were very similar. In each of thyme and clove EOs, derivatization with vanillin-sulphuric acid reagent revealed one major compound (thymol and eugenol) at a retardation factor (hR _F) 48 and 44 (orange and grey zones) respectively, which gave inhibition against Xap isolates ( Figures 2A–F ). Based on the derivatization process, the other EOs were found to be more complex. Among the separated compounds, two in the case of cinnamon at hR _F 41 and 78 and one in the case of tea tree at hR _F 51 displayed characteristic antibacterial activities ( Figures 2A–F ). At the zone close to sample application, where separation is questionable, rosemary gave a strong, while eucalyptus a weak inhibition zone ( Figures 2D–F ). In addition, the rosemary component at hR _F 29 inhibited Xap as well ( Figures 2D–F ). In lemon grass and lemon balm the same two compounds at hR _F 52 and 58 showed antibacterial activities ( Figures 2G–I ). Furthermore, three inhibition zones at hR _F 23, 29, and 80 appeared in citronella grass ( Figures 2G–I ).

The percentage composition of thyme, cinnamon, clove, and eucalyptus EOs was previously determined in our laboratory by GC demonstrating that the main components are thymol (49.9%), trans-cinnamaldehyde (73.2%), eugenol (83.7%), and eucalyptol (84.2%), respectively (Horváth et al., 2013). Thus, not surprisingly, analyzing the compounds in the antibacterial DB zones of these EOs by SPME-GC/MS, thymol (hR _F 48), trans-cinnamaldehyde (hR _F 41), and eugenol (hR _F 44) were identified, respectively. In tea tree EO, also the main component terpinen-4-ol (41.3%) was determined in the inhibition zone. The major rosemary components cineole (41.9%) and camphor (18.5%) did not exhibit inhibitory activities, however, borneol (7.7%) was recognized as responsible for the antibacterial effect at hR _F 29 ( Figures 2D–F ). Neral (cis-citral) and geranial (trans-citral) were presented in the inhibition zones at hR _F 52 and 58, respectively ( Figures 2G–I ), which were the constituents of lemon grass and lemon balm EOs with the highest percentage (each between 32% and 39%). Compounds in antibacterial zones of citronella grass EO at hR _F 23 and 80 were identified as nerol and citronellal that have the highest abundance in the oil with 17.3% and 37.6%, respectively ( Figures 2G–I ).

The antibacterial activity of the compounds identified by the combination of HPTLC-Xap assays and SPME-GC/MS was assessed also by MIC ( Table 1 ). The anti-Xap effect of all compounds was confirmed (with MIC between 50 and 400 µg/mL or nL/mL), except for terpinen-4-ol that did not inhibit the bacterial growth in the applied concentrations. The two Xap isolates showed similar sensitivity to EO components, but citral and borneol were more effective on XapG2 than on Xap3, while the Xap3 isolate was more sensitive to eugenol compared to XapG2. The highest inhibitory activity was attributed to thymol with a MIC of 50 µg/mL.

Importantly, results of relevant studies on antibacterial effects of EOs are difficult to compare, not only because of differences in applied methods, but also because of the variable composition of EOs, influenced by geographical, climatic, edaphic and seasonal factors (Ložienė and Venskutonis, 2005; Aboukhalid et al., 2017; Tsiftsoglou et al., 2023). Regarding Xap, our results enable comparing antimicrobial activities of lemon grass and eucalyptus EOs to literature data. The Xap isolates tested in this study (Xap3, XapG2) showed higher sensitivity (with MIC values of 125 and 250 µg/mL) to lemon grass EO than the Xap strain examined by Zhao et al. (2011) (MIC 500 µL/L). Montesinos et al. (2023) reported a reduced susceptibility of Xap (MIC 10 – 30 µl/mL) and other species such as X. fragariae and X. axonopodis pv. vesicatoria (MIC < 5 and 10 – 30 µl/mL, respectively) to eucalyptus EO, as compared to our observations, however they used a different method, the agar incorporation test. The effectiveness of other EOs on Xap has been previously also revealed; antibacterial effects of Thymbra sintenisii and Satureja cuneifolia EOs were reported with MIC values of 600 ppm and 800 ppm, respectively (Biavati et al., 2004). Furthermore, Mahmoudi et al. (2010) investigated the antibacterial activities of fourteen EOs and four plant extracts on Xap using the agar diffusion method and they have found peppermint, cumin, slender ziziphora, bishop’s weed, common sage and neem tree to be the most effective.

Effects of EOs on other Xanthomonads are widely known; Mačionienė et al. (2021) have found that T. vulgaris EO had the strongest antibacterial effect against X. translucens and five tested X. arboricola strains (unindicated pathovars) with 7.81 µg/mL MIC value as revealed by BMA. However, in case of the Hungarian Xap strains, the T. vulgaris EO provided a higher MIC value (125 µg/mL). Kotan et al. (2007) investigated the effect of EOs of other Thymus spp. on the growth of X. axonopodis pv. vesicatoria by the agar diffusion method, and found that the test bacterium was the most sensitive to T. sipyleus rosulans (MIC 25 µg/mL) out of the other Thymus spp. with 200 µg/mL MIC value. Testing the antibacterial efficacy of another thyme species by BMA, the T. spathulifolius EO was effective against X. campestris-A235 strain at a higher concentration (MIC 500 µg/mL) (Sokmen et al., 2004). Gormez et al. (2016) reported remarkable results testing Origanum rotundifolium EO - whose main compounds are carvacrol and thymol - against X. vesicatoria and X. axonopodis pv. campestris with 7.81 and 31.25 µg/mL MIC values, respectively, using BMA. Mirzaei-Najafgholi et al. (2017), applying BMA as well, found that Citrus aurantifolia EO exhibited a higher activity on X. citri ssp. citri, as compared to that of Citrus aurantium EO with 500 and 1300 µg/mL MIC values, respectively. As for the active ingredients of EOs, Kotan et al. (2007) also investigated the effect of thymol. However, the X. axonopodis pv. vesicatoria strain gave a much lower MIC value (3.125 µg/mL) against thymol than the Hungarian Xap strains in our experiments (50 µg/mL) suggesting that the given X. axonopodis pv. vesicatoria strain is more sensitive to the main active compound of thyme. Cleistocalyx operculatus EO also displayed significant antibacterial activity against four Xanthomonads with MIC values of 31.25–125 μg/mL in BMA (Bajpai et al., 2010). Importantly, although the effects of EOs including that of thyme against X. campestris pv. vesicatoria and X. euvesicatoria was investigated by direct bioautography (Horváth et al., 2004; Móricz et al., 2015a; Móricz et al., 2015b), to our knowledge, this method has not yet been introduced against Xap.

The bioactivity and action mechanism of EOs strongly depends on their composition. EOs are constituted of volatile secondary metabolites including mono-, and sesquiterpenes, as well as oxygenated compounds, like alcohols, phenols, aldehydes, and esters (Hyldgaard et al., 2012; Raveau et al., 2020; Angane et al., 2022). Terpenes of EOs, such as cinnamon or clove EO, are able to disrupt and penetrate the lipid structure of the cell wall and the bacterial cell membrane, leading to protein denaturation and cell membrane destruction, cytoplasmic leakage, pH decrease, loss of DNA transcription, protein synthesis and enzyme activity, cell lysis, and ultimately cell death (Oussalah et al., 2006; Raybaudi-Massilia et al., 2006). In addition, cinnamon EO and its components have been showed to inhibit amino acid decarboxylases in Gram-negative bacteria (Wendakoon and Sakaguchi, 1995). Similar to other phenolic compounds, thymol or eugenol is assumed to damage the structure and function of the cytoplasmic membrane, which disrupts the proton motive force, the electron flow, and active transport causing coagulation of cell contents (Sikkema et al., 1995). Terpenoids of lemon grass also permeabilize the cell membrane (Hyldgaard et al., 2012; Nguefack et al., 2012).

In fact, the antimicrobial activity of EOs is assigned to a number of small terpenoids and phenol compounds (e.g. thymol, carvacrol, eugenol, cinnamaldehyde), which display high antibacterial activities also in pure form (Conner, 1993; Didry et al., 1993; Horváth et al., 2004). However, the efficacy of EOs may not exclusively depend on the ratio of the main active compounds, but also on interactions between active compounds and their minor constituents (Hyldgaard et al., 2012). In such multicomponent mixtures, the possible occurrence of interactions among the components can result in synergistic, additive, or antagonistic effects (Hakalová et al., 2022; Połeć et al., 2022). Thus, similar to our results obtained with cinnamon, clove, lemon grass, lemon balm, and citronella grass EOs, some studies have demonstrated that whole EOs have greater antibacterial activity than the major components (Mourey and Canillac, 2002; Burt, 2004). This synergism may also underlie the fact that cinnamon EO, which exhibited the highest efficacy in our experiments, had a lower MIC than that of its main active ingredient trans-cinnamaldehyde.

To our knowledge, no EO-based antibacterial product have been registered in the EU so far, however, three other types of biopesticides are available; the Mevalone^® biofungicide containing geraniol, eugenol, thymol against Botrytis cinerea in wine grapes, the Bioxeda^® post-harvest fungicide with clove EO as an active ingredient against Gloeosporium spp. and Penicillium spp. on apples, peaches and pears and PREV-AM^® containing orange EO to control whiteflies on tomatoes and zucchini (EC, 2023). In addition, it is important to mention that an EO-based biopesticide against Xap is already available in the USA: Guarda^® with the active ingredient thyme EO formulated by HOLDit^® technology is recommended against Xap besides many fungal and bacterial pathogens (BioSafe Systems, 2023). Applications of EOs as alternative biopesticides under field conditions are confronted by the loss of efficacy or performance due to their volatility, instability, low solubility in water, different viscosity, and aptitude for oxidation (Pavoni et al., 2020). Therefore, EOs require appropriate formulation to preserve their bioactivity and to extend and control their release time. Encapsulation of pesticides is usually ensured by solid nanoparticulate or liquid nanoemulsion systems with the aid of natural polymers (e.g. polysaccharides), wettable powders or granules, surfactants, stabilizers, liposomes and solvents (Benita, 2006; Borges et al., 2018; Pavoni et al., 2020). Such nanocarrier systems stabilize the EOs, along with improving bioavailability and amplifying antimicrobial efficacy (Anwer et al., 2014; Micucci et al., 2020; Wen et al., 2021). Nanoencapsulation of thyme and cinnamon EOs and their main constituents (thymol and cinnamaldehyde, respectively) in nanocarriers were found to be a promising approach for development of biopesticides (Falleh et al., 2021; Sousa et al., 2021; Abdelhamed et al., 2022; Alsakhawy et al., 2022). In comparison to pure EO, nanosponges of cinnamon EO prepared with ethyl cellulose and polyvinyl alcohol had significantly higher antibacterial effects against Staphylococcus aureus (Kaur et al., 2020). Similarly, a nanostructured lipid carrier cinnamon gel had about 10 times higher antibacterial efficacy against P. aeruginosa than the EO alone (Wen et al., 2021), and the effective concentration of cinnamaldehyde to control plant disease caused by Pseudomonas syringae pv. pisi became 90,000-fold lower by a formulation with silica nanoparticles (Bravo Cadena et al., 2018). Furthermore, the nanoemulsion with Thymus daenensis EO, an abundant source of thymol and carvacrol, revealed enhanced antibacterial activity against E. coli compared to the pure EO (Moghimi et al., 2016). In summary, the advanced technology formulation that enables increased stability, efficiency and water solubility could be the key for the widespread application of EOs as antibacterial plant protection agents (Das et al., 2020; López et al., 2021; Shankar et al., 2022; Singh and Pulikkal, 2022).

To our knowledge, this is the first report on the antibacterial activity of these EOs (except for lemon grass and eucalyptus) and their major constituents against Xap. Moreover, the HPTLC-Xap (DB) method was introduced to identify anti-Xap EOs’ ingredients. Our results suggest that out of the ten EOs tested, the most effective ones - cinnamon, thyme, clove and lemon grass - have the potential to be promising candidates for alternative biopesticides to control the plant disease caused by Xap.

The original contributions presented in the study are included in the article/supplementary materials. Further inquiries can be directed to the corresponding authors.

ÁMM and IS: conceptualization, methodology and supervision; JKN, ÁMM, AB, ÁA, and IS: Investigation and data analysis; JKN, ÁMM, and IS: writing - review and editing; ÁMM: visualization, funding, project administration. All authors contributed to the article and approved the submitted version.