Evaluation of the Effect of Four Bioactive Compounds in Combination with Chemical Product against Two Spider Mites Tetranychus urticae and Eutetranychus orientalis(Acari: Tetranychidae)

Currently, pests control using chemical acaricides constitutes worries for ecologists and health care people as these chemical products create damage to the ecosystem as well as the development of spider mites resistance. Such concerns request deep and rapid feedback by looking for new alternative and eco-friendly methods. In recent years, a new field is evolving in the use of essential oils in pest management practices. Essential oils have been considered as potential pest management agents, because they demonstrate to have a broad range of bioactivity, possess contact, and fumigant toxicity. In addition, the major advantages of many plant-based acaricides lie in their low toxicity to agroecosystems. Botanical acaricides composed of essential oils may prove to be a good choice for the more persistent synthetic acaricides. In this study, the acaricidal effect of four plant-derived essential oils against adults of the two important crop pests, Tetranychus urticae (Koch) 1836 and Eutetranychus orientalis (Klein) 1936 are studied. The fumigant toxicity revealed that all the essential oils tested Mentha pulegium L., Lavandula stoechas L., Rosmarinus officinalis L., and Origanum compactum Benth (Lamiaceae family) displayed an acaricidal effect. At the highest dose (625 µl/ml), mortalities recorded were found between 91 and 98% and 92 and 99% at 24 and 48 h, respectively, for T. urticae, and between 90 and 98% and 94 and 99% at 24 and 48 h, respectively, for E. orientalis. The M. pulegium L. essential oil represents the highest activity against E. orientalis and T. urticae. For the binary combination between the EOs (essential oils) and the acaricide based on the active ingredient acequinocyl, the results showed that the mixture of O. compactum EO (essential oil) + acequinocyl exhibited an important acaricidal effect on T. urticae and E. orientalis with 99% at 24 h and 100% at 48 h of mortality, followed by M. pulegium EO + acequinocyl with 92% at 24 h and 95% at 48 h for T. urticae as well as 99% at 24 h and 100% at 48 h for E. orientalis of mortality. Whereas, the mixture of L. stoechas EO + acequinocyl presented the lowest activity against T. urticae and E. orientalis with 82–87% at 24 h and 86–90% at 48 h, respectively. The mixtures (M. pulegium EO + acequinocyl, R. officinalis EO + acequinocyl, and O. compactum EO + acequinocyl) exerted a high acaricidal effect against E. orientalis. These promising results could help to develop botanical pesticides that could be used in integrated pest management.


Introduction
Spider mites including Tetranychus urticae (Koch) 1836 and Eutetranychus orientalis (Klein) 1936 are a major pest of citrus in many countries including Morocco [1,2]. ese two pests cause considerable losses to citrus crops [3,4]. In recent years, these pests have become of great economic importance [5,6], by sucking the cellular juice of the leaves causing yellowing, necrosis, and leaf fall which weakens the plant and reduces the photosynthetic capacity [7]. eir control is often provided by intensive and preventive chemical control [8,9]. In Morocco, the Acequinocyl, Abamectin, Malathion, Spirodiclofen, Bifenazate, Fenpyroximate, Fenazaquin, Tebufenpyrad, Propargite, Milbemectine, Bifenthrine, Dicofol, Clofentezine, Maltodextrine, and Pyridaben are the most acaricides employed to control spider mites including T. urticae and E. orientalis under greenhouses and field [1,10]. Despite advantages such as easy use, the decrease of pests numbers, and their immediate action when compared with natural extracts from plants [11].
is approach has considerable limitations; the examination of the spectrum activity of the functional components used throughout the globe demonstrates that 72% of insecticides, 28% of fungicides, and 46% of acaricides are globally toxic toward auxiliary arthropods and public health [12,13].
Acequinocyl is a naphthoquinone compound discovered in the 1970s by DuPont [14]. It is a proacaricide that breaks down into the active metabolite, a deacetylated product. A mechanistic study showed that the deacetylated metabolite of acequinocyl inhibits respiration in mitochondria at the ubiquinol oxidation site (Q0) of complex III of the electron transfer chain [15,16]. e control by Mentha pulegium, Lavandula stoechas, Rosmarinus officinalis, and Origanum compactum (Lamiaceae family) can be used as alternative approaches for control. Essential oils include a plentiful interest for researchers who always try through their scientific studies to discover the great potential of these substances as anti-inflammatory, antioxidant, antimicrobial, and AC agents [17,18]. Plants from the Lamiaceae family including M. pulegium, L. stoechas, R. officinalis, and O. compactum are well known for their acaricidal dynamic biological activities and many reports have demonstrated the existence of different compounds like terpenes, iridoids, flavonoids, and phenolic constituents the species of this family [19,20]. Moreover, it had been conveyed that the two Lamiaceae plants, M. pulegium and R. officinalis have multiple significant acaricidal activities against spider mites [21,22]. M. pulegium possesses several acaricidal properties; this aromatic plant has demonstrated antioxidant and anticholinesterase, anthelmintic, antimicrobial, insecticidal, and acaricidal effects [23][24][25]. Similarly, R. officinalis is well known for its beneficial effect as a medicinal mechanism and it also showed an acaricidal potential against pests [26]. e objective of this work was to evaluate the acaricidal effect of four essential oils derived from M. pulegium, L. stoechas, R. officinalis, and O. compactum, alone and in combination with a chemical acaricide based on acequinocyl, against two spider mites, T. urticae and E. orientalis.

Plant Material and Extraction of Essential Oils.
Samples of Lavandula stoechas, Mentha pulegium, and Origanum compactum (Lamiaceae family) were collected between April and June (the flowering stage) in the mountainous area that is located in the rural community of Timezgana (Northeastern Morocco, 34°34′48″ North, 4°43′48″ West), at an altitude of approximately 800 m.
Samples of R. officinalis come from the experimental garden of the Faculty of Science and Technology of Fez. e collected plants were identified at the National Agency for Medicinal and Aromatic Plants in Taounate, Morocco. ese products were chosen on the basis of their availability and their effectiveness as a natural acaricide.

Chromatographic Analysis.
Chemical analysis of all essential oils was realized using gas chromatography coupled with mass spectrometry (GC/MS) and coupled with flame ionization detection (GC-FID). e GC/MS analysis was utilized for identification, while GC-FID analysis was used for the quantification of components.

Gas Chromatography (GC-FID) Analysis.
Gas chromatography analyses for all the samples were realized using a Hewlett-Packard (HP 6890) gas chromatograph equipped with an HP-5 capillary column (30 m × 0.25 mm, film thickness of 0.25 µm), FID a detector, and an injector fixed at 275°C. e oven temperature was at 50°C for 5 min and then increased to 250°C at 4°C/min. e nitrogen (1.8 ml/min) was utilized as carrier gas. e samples were diluted to 1/50 in methanol and the injected volume was 1 µl using a split mode ratio: 1/50, flow: 72.1 ml/min. e relative proportions of the EOs components were reported as percentages determined using peak-area normalization. e retention indices (RI) on HP-5 MS column, were identified using homologous series of (C8-C28) alkanes.

GC/MS Analysis.
Chemical analysis was carried out using a Hewlett-Packard gas chromatograph (HP 6890) coupled with a mass spectrometer (HP 5973). e column utilized was HP-5MS (cross-linked 5% PHME siloxane; 30 m × 0.25 mm, film thickness 0.25 µm). e column temperature was set at 50°C and increased to 250°C at a rate of 2°C/min. e carrier gas is helium at 1.5 ml/min, using a split mode ratio: 1/74.7, flow rate: 112 ml/min. MS identities of components were confirmed using the NIST 98 spectral library. e ion source temperature of 230°C with ionization voltages of 70 eV and a scan mass range of 35-450 m/z. e identification of the components was also confirmed by comparing the elution order of the compounds with the relative retention indices reported in the literature.

Spider Mites.
e adult spider mites of T. urticae and E. orientalis (Acarina: Tetranychidae) were collected from a farmer in Fez city 34°02′36″N, 05°00′12″W planted by (Citrus aurantium L., 1753) not previously treated with chemical products (Acaricides). e collected leaves were transferred directly into referenced polyethylene bags to the laboratory for inspection. T. urticae and E. orientalis spider mites on each leaf were determined and counted on both leaf surfaces using a binocular microscope before and after the addition of the tested products. ereafter, 20 adults of each Tetranychidae species (T. urticae and E. orientalis) survived in citrus leaves were placed in glass petri dishes (90 mm × 20 mm). Disks of filter papers that were previously attached to the interior surface of the petri dish cover were impregnated with 10 µl of each concentration. DMSO (0.01%) was used as a negative control. Five replications of treatments and controls were carried out. e toxicity was evaluated after 24 and 48 h of exposure, and the mortality was calculated using the formula of Abbott (1) %Mortality Corrected � %Mortality Observed − %Mortality Control 100 − %Mortality Control × 100.

Statistical Treatment.
Mortality percentages were analyzed by the SPSS software (IBM SPSS Statistics 25.0). e obtained results were also examined with OriginPro 2021 software to determine the significant effect between mean values using the Tukey test (a probability of p ≤ 0.05 is regarded as statistically significant). e different principal component analysis was carried out by using the JMP Pro 14. e LD 50 values were estimated using a probit regression analysis (with SPSS software) according to Finney's mathematical methods [27].

e Effect of the Main Compounds of the Four Tested EOs and
eir Acaricidal Activity. e first principal component analysis (PCA) allowed identifying the existence of correlations (positive and negative) between the major compounds of the four essential oils and the acaricidal activity against E. orientalis and T. urticae in 24 and 48 h. e correlations of the different parameters studied are shown in a correlation matrix (Table 1). e loading plot (Figure 4(a)) and the correlation matrix (Table 3) (Figure 4(b)) showed that essential oils of L. stoechas and R. officinalis are dominated by the highest amount of 1,8-cineole, α-pinene, borneol, and fenchone compounds and they require a high concentration to eliminate E. orientalis. However, O. compactum essential oil is presented with a high amount of thymol and α-terpinene compounds. e M. pulegium was characterized by having the highest amount of r (+)-pulegone and carvone compounds and they require lowest concentration to eliminate E. orientalis.
Based on the result from Figures 4(a) and 4(b), it can be concluded that M. pulegium essential oil exhibited the best acaricide activity of the four essential oils. In fact, this can be explained by a high amount of r(+)-pulegone and carvone compounds in M. pulegium essential oil (74.03 and 5.45%, respectively) compared with L. stoechas, R. officinalis, and O. compactum (the absence of these compounds), but the contributions of others compounds should also be noted. e effect of synergy could also be the origin of this activity.      Evidence-Based Complementary and Alternative Medicine PC1 and PC2 shows that individuals were separated into three main groups. ese results indicated that the concentration has an effect on the acaricidal activity, as previously shown by ANOVA. e first group consisted of C1 (39.06 µl/ml) and C2 (78.125 µl/ml), or the low concentration. e second group is composed of C4 (312.5 µl/ml) and C5 (625 µl/ml), or the high concentration. e third groups were C3 (156.25 µl/ml), or the medium concentration. is graph indicated that when the concentration was high, the acaricidal activity against E. orientalis and T. urticae was elevated, and when the concentration was low, the acaricidal activity against E. orientalis and T. urticae was decreased. e biplot ( Figure 5(b)) showed that the acaricidal activity against E. orientalis and T. urticae improved when the M. pulegium essential oil was used. In contrast, the L. stoechas and O. compactum essential oils revealed a medium acaricidal activity against T. urticae. In addition, the R. officinalis showed a medium acaricidal activity against E. orientalis.

e Effect of the Four Mixtures Between Acaricide and Tested EOs and eir Acaricidal Activity.
e third PCA was enabled to determine the similar mixtures that have the same acaricidal activity between the four mixtures studied (M. pulegium EO + acaricide, R. officinalis EO + acaricide, O. compactum EO + acaricide, and L. stoechas EO + acaricide). e biplot ( Figure 6) illustrated that the individuals (mixtures) were divided into three main groups. e first group was the mixture of M. pulegium EO + acaricide and R. officinalis EO + acaricide. e second group was the mixture of O. compactum EO + acaricide. e third group was L. stoechas EO + acaricide. e mixture of (a) M. pulegium EO + acaricide and (b) R. officinalis EO + acaricide showed the same acaricidal activity, a medium effect against the T. urticae and a low effect against E. orientalis.
is graph also indicated that the acaricidal activity has been improved with the mixture of O. compactum EO + acaricide (synergetic effect), while the mixture of L. stoechas EO + acaricide presented a low acaricidal activity against the E. orientalis and T. urticae (antagonistic effect).

3.5.
e Hierarchical Cluster Analysis. e hierarchical cluster analysis (HCA) allowed us to better classify the studied samples (the four essential oils) according to the  Evidence-Based Complementary and Alternative Medicine acaricidal activity against the E. orientalis and T. urticae (Figure 7). As a confirmation of the third PCA, the individuals were divided into three main clusters: cluster 1 (M. pulegium EO + acaricide and R. officinalis EO + acaricide) characterized by a high acaricidal activity against T. urticae, while it was characterized by a low acaricidal activity against the E. orientalis. However, cluster 2 (O. compactum EO + acaricide) was characterized by a high acaricidal activity against the E. orientalis and T. urticae. Cluster 3 (L. stoechas EO + acaricide) was featured by a low acaricidal activity against the E. orientalis and T. urticae. Both the PCA and HCA concluded that the acaricidal activity against the E. orientalis and T. urticae were significantly influenced by adding the acaricide to essential oils. A synergetic effect with O. compactum EO + acaricide and an antagonistic effect with L. stoechas EO + acaricide.

Discussion
In commercial orchards, the protection against pests is currently assured by intensive and preventive chemical products. Despite advantages such as rapid action in reducing the number of pests and their ease of use compared with natural plant extracts. [20], this strategy has many limitations; the examination of the action spectrum of the active components used throughout the world reveals that 46% of acaricides are globally toxic toward auxiliary arthropods and public health [1,28]. e extracts of plants are remarkably rich in toxins and inhibitors and can be the source of many insecticidal and acaricidal substances exploitable in the control of pests [29,30]. On the contrary, the problem of volatility and the high cost causes several limitations of their usage [31], e essential oils lose their force against environmental conditions. Nanoformulation is a method that may preserve the pesticide performance of plant essential oils [31,32]. Phytochemical products are environmentally friendly, target-specificity, decreased the number of applications, higher acceptability, suitability for rural areas, low cost, biodegradable, easy preparation, and universally accepted. Botanicals are used as an alternative to synthetic acaricides and have been projected as a tool in the future for spider mites control agents, which are shown to function as highly polyphagous herbivore and primary agricultural pests in the world that provokes hard damage to crops.
In this study, M. pulegium, L. stoechas, R. officinalis, and O. compactum (Lamiaceae family) EOs showed an acaricidal potential toward adults of the two crop pests, T. urticae and E. orientalis. Indeed, the mortality varied according to the EO tested, the concentrations used, and the mite species.
R. officinalis was often effective against agricultural crop pests including tetranychid mites [22,33]. e essential oil of R. officinalis and mixtures of its main constituents gave good results in the control of T. urticae Koch, on bean and tomato plants [34].
ymol, a hydrophobic compound, constitutes the major monoterpene in O. compactum; it can penetrate the compartment membrane, which diminishes its impermeability, this mechanism stimulates the entry of other components into the cytoplasm [35,36]. ere is also evidence in  the literature that thymol and eugenol have a neurotoxic effect on arthropods, with different mechanisms of action. ymol can bind to GABA receptors located in the membrane of post-synaptic neurons and disrupt the functioning of synapses [37] under laboratory conditions. A study shows that the essential oils of O. compactum is often toxic against Rhyzopertha dominica and Sitophilus oryzae Linnaeus, 1763 [38]. is was confirmed in our study against T. urticae and E. orientalis. O. compactum significantly inhibited oviposition of T. urticae; it was also toxic to females of this pest. Inhibition of fecundity exceeding 80% [39]. e essential oil of M. pulegium was very toxic against the strawberry spider mite Tetranychus turkestani Ugarov and Nikolskii, 1937 (Acari: Tetranychidae) [19].
e same results in another study were observed on T. urticae, a serious pest of many agricultural crops. e phytotoxicity of M. pulegium was significant against this pest after 24 h [40,41]. e statistical analysis (first PCA) permed to conclude that the acaricidal activity depends on the major compounds of the essential oils tested. A strong correlation was identified between the LD 50 , dihydrocarvone and carvone. us, when the quantity of dihydrocarvone and carvone increased, the acaricidal activity increased. Several studies reported that the principal compound present in the essential oils of thyme and oregano increases (thujone 65.78% and carvone 59.35%) the acaricidal activities against spider mite Tetranychus cinnabarinus Boisd are more important [42]. In another study, 28 monoterpenes including monoterpene hydrocarbons and oxygenated monoterpenes (camphor, 3-carene, carvone, 1,8-cineole, citronellal, β-citronellene, dihydrocarvone, and α-terpineol) of essential oils obtained from different plant species were tested against adults of Sitophilus zeamais Motschulsky under laboratory conditions. e results show that most of the monoterpenes with high concentrations have significantly insecticidal and acaricidal effects on the tested pests [43,44].
e second PCA showed that M. pulegium was found to be more effective, followed by O. compactum EO and L. stoechas. EOs acquired from multiple plants have already been tested for their potential fumigant toward pests species (Choi et al., 2006) [42]. As an example, [45] examined the fumigant toxicity of L. stoechas EOs against three spider mites pests attacking stored products (Lasioderma serricorne Fabricius 1792, R. dominica Fabricius 1792, and Tribolium castaneum Herbst, 1797), the findings of this analysis showed that this EO had strong acaricidal properties with the high rates of mortality were obtained after 24 and 48 h of fumigation. L. stoechas was also tested for its acaricidal action against Tetranychus cinnabarinus Koch, 1836, one of the most important pests, the registered LC 50 and LC 90 values were in the range of 2.92 and 13.0 µg/ml, respectively [42]. Author [35] also recorded an effective acaricidal effect of L. stoechas against Orgyia trigotephras Boisduval, 1828 (Lepidoptera, Lymantriidae). e third PCA and HCA illustrated that the activity of O. compactum EO has been ameliorated when it is mixed with the acaricide (synergetic effect). e R. officinalis and M. pulegium remain have the same activity when they were mixed with the acaricide. However, the activity of L. stoechas reduced when it was mixed with the acaricide (antagonistic effect). Several studies confirm that the mixture of several products often triggers an antagonistic effect or a synergistic effect and all depends on the obtained combination [39,46,47].
Moreover, the fumigant toxicity for EOs mixtures with the chemical acaricides revealed encouraging results where the mortality reached more than 95% for both spider mites. e search for synergistic mixtures aims to reduce the concentrations needed by increasing the biological activity against the target organism. is reduction of concentration may also result in decreased toxicological and environmental risks, and lower production cost [48,49].

Conclusions
is scientific investigation highlighted the acaricidal effect of four essential oils against two important spider mites involved in agriculture damage. e obtained results proved the acaricidal potential of the tested oils and showed the efficacy of the binary mixtures with an acaricidal product reaching the mortality to 100%. ese plant-based natural products can be applied in large field applications using low concentrations (based on the LD 50 values) and minimizing the production cost; they since could be involved in pest management programs as a new tool that could solve pest problems reducing risks to humans and the environment. eir inclusion in an integrated pest management program can contribute greatly to the achievement of the acaricide reduction objectives set by several countries and organizations. In this context, responsible agricultural organizations should actively support the development and implementation of essential integrated pest protection programs. Further studies are needed to test the toxicity of these natural products.

Data Availability
All related data are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.