First Report on the Synergistic Interaction between Essential Oils against the Pinewood Nematode Bursaphelenchus xylophilus

Control of the pinewood nematode (PWN), the causal agent of pine wilt disease, can be achieved through the trunk injection of nematicides; however, many pesticides have been linked to environmental and human health concerns. Essential oils (EOs) are suitable alternatives due to their biodegradability and low toxicity to mammals. These complex mixtures of plant volatiles often display multiple biological activities and synergistic interactions between their compounds. The present work profiled the toxicity of eight EOs against the PWN in comparison to their 1:1 mixtures, to screen for successful synergistic interactions. Additionally, the main compounds of the most synergistic mixtures were characterized for their predicted environmental fate and toxicity to mammals in comparison to emamectin benzoate, a commercial nematicide used against PWN. The mixtures of Cymbopogon citratus with Mentha piperita and of Foeniculum vulgare with Satureja montana EOs showed the highest activities, with half-maximal effective concentrations (EC50) of 0.09 and 0.05 µL/mL, respectively. For these, complete PWN mortality was reached after only ca. 15 min or 2 h of direct contact, respectively. Their major compounds had a higher predicted affinity to air and water environmental compartments and are reported to have very low toxicity to mammals, with low acute oral and dermal toxicities. In comparison, emamectin benzoate showed lower nematicidal activity, a higher affinity to the soil and sediments environmental compartments and higher reported oral and dermal toxicity to mammals. Overall, uncovering synergistic activities in combinations of EOs from plants of different families may prove to be a source of biopesticides with optimized toxicity against PWNs.


Nematicidal Activity of EOs and EO Mixtures
The nematicidal activity of EOs and their 1:1 mixtures was compared to that of the commercial nematicide Pursue ® , currently used in Europe in the pest management of PWN. Pursue was assayed at 1 mg of emamectin benzoate (its active compound) per mL of methanol or ultrapure water, and induced strong mortalities, namely, 89.6 ± 0.9 and 88.9 ± 0.4%, respectively. In comparison, the EO of O. vulgare showed a similar strong activity (88.8 ± 0.3%) while that of E. globulus, M. piperita, R. officinalis and S. officinalis were mostly inactive when tested solely (bold values in Table 2). The EOs of C. citratus, F. vulgare or S. montana induced complete mortality (100%) at 1 µL/mL and were tested at lower concentrations. For these EOs, the half-maximal effective concentration (EC 50 ) values were determined to characterize their toxicological strength ( Table 2). Table 2. Nematicidal activity of the essential oils (EOs), on diagonal, and EO mixtures, below diagonal, expressed through their half-maximal effective concentration (EC 50 , in µL/mL) or corrected mortality (%) values at 1 µL/mL. Satureja montana EO had the highest activity (0.15 ± 0.00 µL/mL) followed by C. citratus EO (0.29 ± 0.01 µL/mL) and lastly the EO of F. vulgare (0.51 ± 0.10 µL/mL) ( Table 2).
The 1:1 mixtures of EOs induced different toxicities on PWNs, with some showing additive interactions while others showed synergistic or antagonistic interactions (Table 2). For C. citratus EO, an antagonistic interaction was observed for its combination with the EO of E. globulus, not reaching complete activity at 1 µL/mL, while combinations with the remaining EOs induced EC 50 values lower than that of their constituent EOs tested solely. The mixtures of C. citratus with R. officinalis (EC 50 = 0.19 µL/mL) or S. officinalis (EC 50 = 0.14 µL/mL) EOs resulted in an increase in activity; however, only its combination with M. piperita EO could be considered strongly synergistic (EC 50 = 0.09 µL/mL), for inducing a ca. threefold lower EC 50 value than that of C. citratus EO tested alone (Figure 1a). The combination of E. globulus EO with the other EOs was either additive or antagonistic, namely, its combination with the EOs of C. citratus, F. vulgare or S. montana. An antagonistic interaction was found between the EOs of F. vulgare and R. officinalis; however, synergistic interactions were also found, in its combination with O. vulgare (EC 50 = 0.13 µL/mL) or S. montana (EC 50 = 0.05 µL/mL), in which activity was 10-fold lower than the activity obtained for F. vulgare EO and 3-fold lower than the activity obtained for S. montana EO, tested solely ( Figure 1b). Additionally, slightly synergistic interactions were found for the EOs of M. piperita with O. vulgare, and O. vulgare with the EOs of R. officinalis or S. officinalis.
solely. The mixtures of C. citratus with R. officinalis (EC50 = 0.19 µL/mL) or S. officinalis (EC50 = 0.14 µL/mL) EOs resulted in an increase in activity; however, only its combination with M. piperita EO could be considered strongly synergistic (EC50 = 0.09 µL/mL), for inducing a ca. threefold lower EC50 value than that of C. citratus EO tested alone (Figure 1a). The combination of E. globulus EO with the other EOs was either additive or antagonistic, namely, its combination with the EOs of C. citratus, F. vulgare or S. montana. An antagonistic interaction was found between the EOs of F. vulgare and R. officinalis; however, synergistic interactions were also found, in its combination with O. vulgare (EC50 = 0.13 µL/mL) or S. montana (EC50 = 0.05 µL/mL), in which activity was 10-fold lower than the activity obtained for F. vulgare EO and 3-fold lower than the activity obtained for S. montana EO, tested solely ( Figure 1b). Additionally, slightly synergistic interactions were found for the EOs of M. piperita with O. vulgare, and O. vulgare with the EOs of R. officinalis or S. officinalis. For the most successful EO mixtures and respective constitutive EOs, the half-maximal effective time (ET50, in min) values along with the lowest maximal effective time (ET100, in min) and lowest maximal effective concentration (EC100, in µL/mL) values were determined ( Table 3). The fastest-acting EO was that of S. montana with an ET50 of 2.5 min, followed by the mixture of C. citratus with M. piperita EOs, the EO of C. citratus applied solely, the mixture of S. montana with F. vulgare EOs and lastly by the EO of F. vulgare applied alone ( Table 3). The lowest direct contact time required to fully immobilize the PWN population (ET100) was obtained for S. montana EO, followed by the combination of C. citratus with M. piperita EOs, the EO of C. citratus, the combination of S. montana and F. For the most successful EO mixtures and respective constitutive EOs, the half-maximal effective time (ET 50 , in min) values along with the lowest maximal effective time (ET 100 , in min) and lowest maximal effective concentration (EC 100 , in µL/mL) values were determined ( Table 3). The fastest-acting EO was that of S. montana with an ET 50 of 2.5 min, followed by the mixture of C. citratus with M. piperita EOs, the EO of C. citratus applied solely, the mixture of S. montana with F. vulgare EOs and lastly by the EO of F. vulgare applied alone ( Table 3). The lowest direct contact time required to fully immobilize the PWN population (ET 100 ) was obtained for S. montana EO, followed by the combination of C. citratus with M. piperita EOs, the EO of C. citratus, the combination of S. montana and F. vulgare EOs and, finally, F. vulgare EO tested alone. The lowest concentration required to eliminate 100% of the PWN population was found for the combination of S. montana and F. vulgare EOs, followed by S. montana EO, the combination of C. citratus with M. piperita EOs, F. vulgare EO and lastly the EO of C. citratus (Table 3). Table 3. Half-maximal effective time (ET 50 , in min, average ± standard error), lowest maximal effective time (ET 100 , in min; with 95% confidence intervals) and lowest maximal effective concentration (EC 100 , in µL/mL; with 95% confidence intervals) of the most successful essential oils (EO) or EO mixtures.

Potential Environmental Fate and Human Health Impacts of the Main EO Volatiles
To estimate the environmental fate of the most active EO mixtures (EOs of S. montana with F. vulgare and EOs of C. citratus with M. piperita), the predicted environmental distribution (PED) of their main EO compounds (≥5%), namely, trans-anethole, carvacrol, geranial, geraniol, menthone, β-myrcene, neral, pulegone, α-pinene and γ-terpinene, was compared to that of emamectin benzoate (Table 4). Information on the isomers geranial and neral was mainly found for their mixture, designated as citral, which is more commonly found in natural conditions. The predicted environmental fate of each EO compound differed greatly from that of the (hemi)synthetic emamectin benzoate. Due to their volatile nature, EO compounds were predicted to be favorably distributed in the air environmental compartment, with percentages that varied from 19 (citral) to 100% (α-pinene and β-myrcene), with the exception of carvacrol (2%). On the other hand, emamectin benzoate showed a very low predicted affinity to this compartment (ca. 50 parts per million) ( Table 4). For the water environmental compartment, the greatest affinities were predicted for citral (68%), geraniol (65%), trans-anethole (28%), pulegone (27%), carvacrol (23%) and menthone (22%), while for the remaining compounds, the percentages were below 0.5%. The predicted distribution of emamectin benzoate in the soil environmental compartment was the highest of all the analyzed compounds (ca. 98%), followed by carvacrol (73%), pulegone (39%), trans-anethole (30%), geraniol (13%) and citral (12%), with the remaining compounds showing percentages below 6% (Table 4). In the environmental compartment of the sediments, the EO volatiles obtained a consistently lower percentage (between 0.003%, for β-myrcene, and 1.6%, for carvacrol) than emamectin benzoate (2.17%). Table 4. Main compounds of the nematicidal essential oil mixtures (≥5%) determined through gas chromatography-mass spectrometry (GC-MS), their predicted environmental distribution (PED) percentages in the air, water, soil and sediments environmental compartments computed through the Mackay fugacity model [15], and their oral and dermal acute toxicities for mammals (median lethal dose, LD 50 , mg/kg) obtained from PubChem online database [16] and PPDB: the Pesticide Properties Database [17]. To understand the potential safety for human health in the usage of EO mixtures, the oral and dermal toxicity thresholds for mammals were retrieved from online databases and compared to those of emamectin benzoate. The values of lethal doses, LD 50 , mg/kg, were shown to be consistently higher for EO compounds than for the commercial nematicide (Table 4), indicating their lower toxicity for mammals. For acute oral toxicity, LD 50 values varied from 7-, for pulegone, to 108-fold, for citral, higher than the value reported for emamectin benzoate. For acute dermal toxicity, the LD 50 values ranged from >1000 mg/kg, for citral, to >5000 mg/kg, for α-pinene, trans-anethole, geraniol or β-myrcene, which is roughly 2-to 11-fold the LD 50 value reported for emamectin benzoate (439 mg/kg) ( Table 4).

Discussion
The tested EOs showed quantitative and qualitative differences in their volatile compositions, which influenced the nematicidal activity of each EO. The most active EOs, namely, those of C. citratus, F. vulgare and S. montana, were rich in oxygen-containing compounds, namely, trans-anethole, carvacrol, geranial, geraniol and neral; however, compounds without oxygen were also present in high amounts, namely, β-myrcene and α-pinene. This suggests that nematicidal activity was imposed by specific oxygen-containing compounds, i.e., compounds with electronegative elements. This same effect has been reported in a previous work, where the oxygen-containing molecules from EOs with high activity against PWNs were separated from the respective hydrocarbon molecules [18]. In fact, this characteristic appears to be transversal to the majority of reports on the use of EOs against PWNs [12]. Nevertheless, in the present work, the EOs with the lowest activities against PWNs, namely, those of E. globulus, M. piperita, R. officinalis and S. officinalis, also had high amounts of oxygen-containing molecules in their compositions, e.g., 1,8-cineole, menthone, pulegone, α-thujone, β-thujone and verbenone, and, again, the monoterpene hydrocarbons β-myrcene and α-pinene were present in high amounts. In fact, the major oxygen-containing compounds of the EOs with the highest activity had phenol, aldehyde and alcohol functional groups, while the oxygen-containing compounds of the EOs with the lowest activities were cyclic ether and ketones, suggesting that volatiles from the former groups may induce higher activities than the latter against PWNs. A similar observation was previously reported for the screening of commercially acquired monoterpenes against PWNs [19]. In this work [19], the phenols carvacrol and thymol (positional isomers), the alcohols geraniol, nerol (cis and trans isomers), menthol and citronellol and the aldehydes citronellal and citral (geranial and neral) showed higher activities than the tested hydrocarbons, e.g., β-myrcene, α-pinene or β-pinene, or ketones, e.g., menthone, pulegone or carvone. Additionally, the trunk injection nematicide levamisole hydrochloride was tested as a positive control, showing lower activity than the most active monoterpenes. This nematicide has a high affinity to soil and sediment environmental compartments and is reported to have a low LD 50 value for oral toxicity (180 mg/kg), similarly to emamectin benzoate [15][16][17]20].
The EOs of C. citratus have been previously tested against PWNs with good results [11,18,21,22]. In these works, the EO volatile profiling, when performed, showed a composition in major volatiles like the one of the EO used in the present study. The nematicidal activity was very similar to that reported in the present study at the tested concentrations; however, the EC 50 values, when determined, were slightly higher, suggesting that small variations in compound amounts may cause variability in the activity against PWNs. Additionally, a visual assessment of nematode mortality under a microscope can be easily prone to variability being heavily dependent on the observers and their experience.
For F. vulgare, the EOs tested in previous works showed volatile compositions similar to that of the one used in the present work but induced only low-to-moderate mortalities in PWNs, at 2 µL/mL [18,22,23]. The EO that showed the highest activity (a 66% corrected mortality at 2 µL/mL) was tested with methanol as a solubilizer, similarly to the present work, which might suggest an influence of the solubilizer compound on the activity of some EOs in direct-contact bioassays. In fact, a previous work compared the suitability of two solubilizer compounds with different chemical characteristics, Triton X-100, a nonionic detergent-type surfactant, and acetone, a polar aprotic organic solvent, and found that, for some EOs, the solubilizer strongly influenced their activity against PWNs, while in others, only slight differences were reported [23]. This indicates that the combination of EO compounds with a solubilizing agent can have a strong influence on the observed nematicidal activity of the EO. The EOs of S. montana assayed in previous works showed quantitative variations in their volatile composition which appeared to influence the activity against the PWN [18,22,23]. In bioassays at 2 and 1 µL/mL, S. montana EOs showed complete mortality (100%); however, at 0.5 µL/mL, while one EO (with 64% of carvacrol and 18% of γ-terpinene) showed 60% mortality; the other EO (with 40% of carvacrol, 20% of p-cymene and 15% of thymol, a positional isomer of carvacrol) showed only 25% corrected mortality, indicating that the amounts of carvacrol, γ-terpinene and/or p-cymene can influence activity against PWNs.
The synergistic interactions in mixtures of EOs were determined for the first time for the PWN. Additive, synergistic, and antagonistic interactions were detected in varying degrees. Combinations featuring the EO of E. globulus showed either additive or antagonistic interactions, indicating that its volatiles, mainly 1,8-cineole and α-pinene, may have some inhibitory activity on other nematicidal EO compounds, against PWNs. In fact, in a study on the interactions of EO compounds with activity against the yellow fever mosquito Aedes aegypti, responsible for the transmission of serious human diseases, 1,8-cineole (eucalyptol) showed mainly antagonistic interactions with other monoterpenes, e.g., carvone and limonene, but synergistic interactions with α-pinene, while α-pinene showed either antagonistic or no interaction with other terpenes [24]. However, in a study on the interactions of several characteristic EO compounds against Spodoptera littoralis larvae, most synergistic interactions were attributed to six compounds, among them, trans-anethole, γ-terpinene and p-cymene [25]. Most notably, synergistic interactions were found for binary mixtures of trans-anethole with carvacrol or γ-terpinene, α-pinene with γ-terpinene and β-myrcene with menthone. In a follow-up study on these EO compounds against Culex quinquefasciatus larvae, strong synergistic interactions were found for the binary mixtures of β-myrcene with menthone and trans-anethole with γ-terpinene, the binary mixtures of carvacrol with trans-anethole or α-pinene showed milder synergistic interactions [26].
In the present work, the strongest synergistic interactions were obtained for the combination of S. montana with F. vulgare EOs, suggesting the existence of this interaction between the main components of each EO, namely, trans-anethole with carvacrol and/or γ-terpinene. In a study analyzing the interactions of EO compounds against the animal parasitic nematode Haemonchus contortus, 1:1 mixtures of carvacrol with cinnamaldehyde, a phenylpropanoid, or thymol, its isomer, showed synergistic interactions; its 1:1 combination with carvone (as well as the combination thymol/carvone) showed an antagonistic interaction; however, its interaction with trans-anethole was only additive [27]. On the other hand, trans-anethole showed a synergistic interaction with carvone but additive with thymol and cinnamaldehyde. In another study, where mixtures of EO compounds were bioassayed against the phytoparasitic nematode Meloidogyne incognita, F. vulgare EO's main compound trans-anethole showed synergistic interactions with carvacrol (at 2:1.25 ratio) or its isomer thymol (at 3:1.25 ratio) [13], suggesting a specificity on synergistic interactions with regard to the nematode group.
Another highly synergistic combination detected in the present study was that of C. citratus with M. piperita EOs. Synergistic interactions have been reported before for C. citratus EO main compounds geranial and neral [28]. In this study, citral was profiled in C. citratus EO with relative amounts of 43% for geranial and 33% for neral. Additionally, its Plants 2023, 12, 2438 9 of 15 combination with 2-undecanone, an aliphatic ketone commonly present in the EO of Ruta graveolens, in a 2:1 ratio resulted in a strong synergistic activity against the phytoparasitic nematode M. incognita, in bioassays in vitro, through direct-contact bioassays, or in vivo, using potted tomato plants in controlled greenhouse conditions. In another study that screened the interaction of EO compounds at different ratios, pulegone, a ketone characteristic of M. piperita EO, in a 1:2 combination with geraniol, commonly found in C. citratus EO, was reported to show only an additive effect, which may indicate a specificity in the ratios of compounds with regard to their nematicidal interactions [13].
In the present study, the time required for complete mortality was determined for the first time in the bioassay of EOs and EO mixtures against PWNs. This parameter is very important for the development of bionematicides since a compromise must be found between the time that the EO takes to exert its effect and its characteristic volatility or biodegradability. Surprisingly, the most successful EOs and their mixtures appear to act quite quickly, enforcing their potential for the development of successful biopesticides. In other studies, the shortest bioassay duration was 4 h, with the EOs of Allium sativum (composed of diallyl sulfide, diallyl disulfide and diallyl trisulfide) showing complete mortality at 0.0625 mg/mL and Allium cepa (composed of propyl trisulfide, propyl disulfide, methyl propyltrisulfide and methyl propyldisulide) showing EC 50 values as low as 0.0121 mg/mL [21,29]. However, lower bioassay time periods were not bioassayed.
Despite the successful activities shown by EO mixtures, their potential use for the innovation of bionematicides also depends on higher safety for the environment and low toxicity to humans, in comparison to the currently used nematicides. Generally, the environmental risk assessment of pesticide compounds mainly depends on combining data on exposure and effects. In the case of exposure to the environment, the Mackay fugacity models are a good approach to predict the affinity of a stressor to the several environmental compartments, using established in silico computations, while for the data on the effects, several online databases offer compilations of data on experimental assays, conducted in standard conditions, that evaluate acute or chronic toxicities. In the present work, as a contribution to this evaluation, the predicted environmental distribution of the main EO compounds as well as their reported acute oral and dermal toxicities were compared to those of the commercial nematicide's active substance. The EO volatiles were mainly predicted to be dispersed to either the atmosphere or water deposits; furthermore, their toxicity to mammals was consistently lower than that of emamectin benzoate. This nematicide has a high affinity with soil and sediments and also higher toxicity to mammals than EO volatiles. In fact, the analyzed EO compounds are currently categorized as flavoring agents, additives that improve aroma or taste and have been approved for safe human consumption [30]. For example, for C. citratus EOs tested on rats, with daily intake over 14 days, only doses above values as high as 1500 mg/kg were seen to exert any functional damage to the stomach and liver [31]. Additionally, being authorized for human consumption is known to greatly facilitate the process of approval for new bionematicides or plant protection products (PPPs), when compared with synthetic compounds [32].
Concerning their environmental safety, although a great affinity was predicted for the air and water environmental compartments, the EO compounds are volatile and highly biodegradable, which leads to very low recalcitrance in the environment. Additionally, citral and geraniol have a higher affinity to the water environmental compartment; however, their reported LC 50 for fish is about 6.1 mg/L and 11.6 mg/L, respectively, a higher value than 0.174 mg/L, reported for emamectin benzoate [16]. The same tendency is reported for other aquatic test model organisms, which shows that the compounds with a higher risk of spreading to the aquatic biota, due to their higher affinity for this compartment, are reportedly less toxic than emamectin benzoate. Concerning the air environmental compartment, ecotoxicological information was found for pollinator insects, for example, the honeybee (Apis mellifera). While emamectin benzoate has a reported LD 50 of 0.036 µg/bee, the monoterpenes carvone or citral have extremely higher values, 106,620 µg/bee and 78,459 µg/bee, respectively, which even makes them potential biopesticides able to control Varroa destructor, a highly damaging ectoparasite of honeybees [16,33]. Ultimately, the identified EO mixtures can offer a safer alternative to conventional nematicides while showing improved nematicidal activity against PWNs.

Chemical Profiling of EOs and EO Mixtures
The chemical composition of EOs and their mixtures was profiled with a Shimadzu GC2010 gas chromatographer coupled to a GC-MS-QP2010 Plus mass spectrometer (Shimadzu, Kyoto, Japan), by injecting 0.1 µL of a sample of each EO diluted (1:1, v/v) in n-hexane (95%, Optima grade for HPLC and GC-MS, Fisher Chemicals, Hampton, NH, USA). The chromatographic separation was performed with a Zebron column ZB-5HT (30 m length, 0.25 mm I.D., 0.25 µm film thickness) (Phenomenex, Torrance, CA, USA). Injections were performed using a split sampling technique (ratio 1:100) with the injector temperature set to 250 • C and a helium (He) flow of 1.5 mL/min. The GC oven temperature program was set to increase from 45 to 175 • C, at 3 • C/min, and then up to 300 • C, at 15 • C/min, with a final isothermal step for 10 min [18]. The mass spectrometer was operated in EI mode (70 eV) and scanned from 40 to 850 m/z. The ion source temperature was set at 240 • C, and the interface temperature was maintained at 280 • C. Peak assignment was performed using the National Institute of Standards and Technology (NIST), Wiley and laboratory-built mass spectra libraries, through AMDIS software (National Institute of Standards and Technology of the US Department of Commerce, Gaithersburg, MD, USA).

In Vitro Culturing of Pinewood Nematodes
To obtain large quantities of pinewood nematodes for the direct-contact bioassays, the isolate Bx0.13.003 was used, which is a reference isolate kept at the Plant Nematology Lab of the National Institute for Agrarian and Veterinary Research (INIAV, I.P.) at Oeiras, Portugal, for research purposes. Bx0.13.003 was isolated from a Pinus pinaster field tree displaying strong PWD symptomatology (N 39 • 43 338 , W 9 • 01 557 ). An internal transcribed spacer (ITS) region sequence was deposited in the GenBank database (NCBI) with the accession number MF611984.1. Larger quantities of this PWN isolate were obtained by culturing in a non-sporulating Botrytis cinerea (de Bary) Whetzel strain in aseptic conditions. For this purpose, axenic cultures of B. cinerea were established on steam-sterilized hydrated certified organic commercial barley grains (Hordeum vulgare L.) (ca. 15 g cereal/15 mL ultrapure water, in 250 mL Erlenmeyer flasks) for 7 to 10 days at 25 ± 1 • C. Fungal mats, obtained after the surface of the cereal was fully colonized, were inoculated with 1 mL of a mixed-life-stage PWN suspension (1000 PWNs/mL) and kept at 25 ± 1 • C in darkness for 7 to 10 days until the fungal mat was consumed. To prevent unwanted microbial contamination that might influence mortality, the nematodes were surface sterilized before the last subculturing with an ethanol solution in ultrapure water (50% v/v) for 5 min [34] and then re-established on axenic mycelial mats. Nematodes were extracted using the modified Baermann funnel technique [35]. Aqueous solutions of PWNs were used for the direct contact assays, for further inoculations, or stored at 11 • C. The assessment of PWN numbers and/or survival rates was performed using an Olympus SZX12 (Tokyo, Japan) stereomicroscope (40×).

Nematicidal Activity of the Essential Oils and EO Mixtures
Direct-contact bioassays were performed in flat-bottom 96-well microtiter plates (Carl Roth GmbH & Co. KG, Karlsruhe, Germany). In each well, 95 µL of an aqueous suspension of mixed-life-stage PWNs (80-100 PWNs) were added to 5 µL of EO stock solution (prepared in HPLC-grade methanol, Fisher Chemicals, Hampton, NH, USA, at 20 µL/mL), to obtain a final EO concentration of 1 µL/mL. Blank wells were added with 5 µL of ultrapure water instead of the EO stock solution, to assess natural PWN mortality, and control wells with 5 µL of methanol, to determine mortality caused by the organic solvent. The microtiter plates were sealed with plastic film to prevent EO volatilization and mixed in an orbital shaker (IKA labortechnik, Staufen, Germany) at 800 cycles/min for 1 min. Plates were covered with aluminum foil to establish complete darkness and maintained for 24 h at 50 r.p.m. in an orbital shaker at 25 ± 1 • C. To determine EO nematicidal strength, live and dead PWNs were counted under a stereomicroscope (40×). To ascertain mortality, physical prodding was used on motionless PWNs to stimulate movement. If no movement was detected PWNs were considered dead. Three separate trials were performed for each sample in a total of 10 bioassays. EOs that showed full mortality were screened at lower EO concentrations (0.5, 0.25, 0.13, 0.06 and 0.03 µL/mL obtained by serial dilutions with a dilution factor of two) to determine toxicity thresholds.
For EO mixtures, stock solutions of combined EOs (2 per mixture) were prepared in methanol as described above. For each combination's stock solution, individual EOs were added for a final concentration of 20 µL/mL each (20 µL/mL of EO #1 and 20 µL/mL of EO #2). Lower EO combination concentrations were obtained by serial dilutions with a dilution factor of two.
To determine the effective time thresholds for activity, the mortality of the most successful EOs and EO mixtures was determined at ca. 5, 30, 240 and 1440 min after application, at the lowest tested concentration that caused complete mortality (100%).

Predicted Environmental Fate of the Main Volatiles of the Most Successful EO Mixtures
The potential environmental fate of the main EO compounds identified in the EO mixtures with the highest nematicidal activity was determined through the predictive equilibrium criterion model suggested by Mackay et al. [15] and compared to that of the synthetic nematicide emamectin benzoate. Supported by this model, predicted environmental distribution (PED) percentages were obtained for each compound in the environmental compartments of air, water, soil and sediments by using the freely available Level I Mackay Fugacity Model beta version 4.31, Trent University, Canada [20]. The model for this level predicts a situation in which a fixed quantity of compound, namely, an illustrative 100,000 kg, is introduced in a closed system, under steady-state and equilibrium conditions, at a temperature of 25 • C. The chemical parameters needed from each compound, namely, molecular mass (g/mol), melting point ( • C), vapor pressure (Pa), solubility in water (mg/L), air-water partition coefficient or Henry's Law constant (Pa.m 3 /mol), n-octanol/water partition coefficient (log value of Kow) and soil organic carbon/water partition coefficient (Koc) were retrieved from the PubChem online database [16] and the PPDB: the Pesticide Properties Database [17] (Table 5).

Data Treatment and Statistical Analysis
Nematode mortality percentages were determined according to the formula: mor-tality% = [(dead PWNs)/(live + dead PWNs)] × 100. For each EO or EO combination concentration, corrected mortality percentages were determined using the formula: corrected mortality% = [(mortality% in treatment − mortality% in control)/(100 − mortality% in control)] × 100. The categorization established by Kong et al. [11] was used to classify the toxicological strength at each concentration, by considering mortality as complete when at 100%, strong when above 80%, moderate between 80 and 61%, weak between 60 and 40%, and low or inactive below 40%.
The determination of the half-maximal effective concentration (EC 50 ) values was performed with Version 2019 of Origin Graphing and Analysis software (OriginLab, Northampton, MA, USA). A nonlinear regression analysis was performed by plotting corrected mortality values along EO or EO combination concentration values, and fitting a dose-response log-logistic equation: y = C + (D − C)/1 + exp {b [log (x) − log (EC 50 )]} [36], where C and D are the lower and upper limits of the sigmoidal dose-response curve, respectively; b is the slope, and EC 50 is the EO concentration which induces a response halfway between the lower and upper limits. The upper (D) and lower (C) limits were set to 0 and 100%, respectively. For the assessment of synergistic and antagonistic interactions, the activities of the EO mixtures were compared to the sum of single EO activities, as described by Pavela [26] and Faraone et al. [37]. For the determination of the half-maximal effective time (ET 50 ) values, the analysis parameters described above were equally applied except for the time values being plotted against the corrected mortality values, at the highest EO or EO mixture concentrations that induced complete mortality (100%). Thus, ET 50 is the time that induces a response halfway between the lower and upper limits. The determination of the lowest maximal effective concentration (EC 100 ) and lowest maximal effective time (ET 100 ) was performed by solving the curve equation to the first y value of 100% mortality.

Conclusions
Essential oils are known to show strong nematicidal activities. Some are even used against plant parasitic nematodes, either whole or using their main compounds, in commercialized nematicidal formulations. Improving their nematicidal activity by combining EOs from plants of different families was tested for the first time for the PWN. When compared to the currently commercialized nematicides, the EO mixtures of Cymbopogon citratus with Mentha piperita and of Foeniculum vulgare with Satureja montana were strongly and quickly active, and they were predicted to volatilize to the atmosphere and have low toxicity for mammals, which turns them into strong candidates for the development of environmentally friendly biopesticides.