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

Authors: Amine Assouguem; Mohammed Kara; Amal Ramzi; Saoussan Annemer; Alicja Kowalczyk; Essam A. Ali; Bushra Abdulkarim Moharram; Abderrahim Lazraq; Abdellah Farah
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

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.


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## Body

## 1. Introduction

Spider mites includingTetranychus urticae (Koch) 1836 and Eutetranychus orientalis (Klein) 1936 are a major pest of citrus in many countries including Morocco [1, 2]. These 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]. Their 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]. This 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].The control byMentha 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 [23–25]. Similarly, R. officinalis is well known for its beneficial effect as a medicinal mechanism and it also showed an acaricidal potential against pests [26]. The 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.


## 2. Materials and Methods

### 2.1. Plant Material and Extraction of Essential Oils

Samples ofLavandula 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 ofR. officinalis come from the experimental garden of the Faculty of Science and Technology of Fez. The collected plants were identified at the National Agency for Medicinal and Aromatic Plants in Taounate, Morocco.These products were chosen on the basis of their availability and their effectiveness as a natural acaricide.


### 2.2. 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). The GC/MS analysis was utilized for identification, while GC-FID analysis was used for the quantification of components.


#### 2.2.1. 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 0.25µm), FID a detector, and an injector fixed at 275°C. The oven temperature was at 50°C for 5 min and then increased to 250°C at 4°C/min. The nitrogen (1.8 ml/min) was utilized as carrier gas. The 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. The relative proportions of the EOs components were reported as percentages determined using peak-area normalization. The retention indices (RI) on HP-5 MS column, were identified using homologous series of (C8–C28) alkanes.


#### 2.2.2. GC/MS Analysis

Chemical analysis was carried out using a Hewlett-Packard gas chromatograph (HP 6890) coupled with a mass spectrometer (HP 5973). The column utilized was HP-5MS (cross-linked 5% PHME siloxane; 30 m × 0.25 mm, film thickness 0.25µm). The column temperature was set at 50°C and increased to 250°C at a rate of 2°C/min. The 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. The ion source temperature of 230°C with ionization voltages of 70 eV and a scan mass range of 35–450 m/z. The identification of the components was also confirmed by comparing the elution order of the compounds with the relative retention indices reported in the literature.


### 2.3. Spider Mites

The adult spider mites ofT. 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). The 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.


### 2.4. Toxicity Test

The acaricidal tests were conducted according to the methodology of Sertkaya et al. (2010) with slight modifications. Firstly, various doses (39.06, 78.125, 156.25, 312.5, and 625µl/L) of each EO and the chemical acaricide (Kanemite, 15%) were prepared. Thereafter, 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. The toxicity was evaluated after 24 and 48 h of exposure, and the mortality was calculated using the formula of Abbott (1)(1)%Mortality Corrected=%Mortality Observed−%Mortality Control100−%Mortality Control×100.


### 2.5. Statistical Treatment

Mortality percentages were analyzed by the SPSS software (IBM SPSS Statistics 25.0). The obtained results were also examined with OriginPro 2021 software to determine the significant effect between mean values using the Tukey test (a probability ofp≤0.05 is regarded as statistically significant). The different principal component analysis was carried out by using the JMP Pro 14. The LD50 values were estimated using a probit regression analysis (with SPSS software) according to Finney’s mathematical methods [27].


## 2.1. Plant Material and Extraction of Essential Oils

Samples ofLavandula 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 ofR. officinalis come from the experimental garden of the Faculty of Science and Technology of Fez. The collected plants were identified at the National Agency for Medicinal and Aromatic Plants in Taounate, Morocco.These products were chosen on the basis of their availability and their effectiveness as a natural acaricide.


## 2.2. 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). The GC/MS analysis was utilized for identification, while GC-FID analysis was used for the quantification of components.


### 2.2.1. 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 0.25µm), FID a detector, and an injector fixed at 275°C. The oven temperature was at 50°C for 5 min and then increased to 250°C at 4°C/min. The nitrogen (1.8 ml/min) was utilized as carrier gas. The 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. The relative proportions of the EOs components were reported as percentages determined using peak-area normalization. The retention indices (RI) on HP-5 MS column, were identified using homologous series of (C8–C28) alkanes.


### 2.2.2. GC/MS Analysis

Chemical analysis was carried out using a Hewlett-Packard gas chromatograph (HP 6890) coupled with a mass spectrometer (HP 5973). The column utilized was HP-5MS (cross-linked 5% PHME siloxane; 30 m × 0.25 mm, film thickness 0.25µm). The column temperature was set at 50°C and increased to 250°C at a rate of 2°C/min. The 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. The ion source temperature of 230°C with ionization voltages of 70 eV and a scan mass range of 35–450 m/z. The identification of the components was also confirmed by comparing the elution order of the compounds with the relative retention indices reported in the literature.


## 2.2.1. 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 0.25µm), FID a detector, and an injector fixed at 275°C. The oven temperature was at 50°C for 5 min and then increased to 250°C at 4°C/min. The nitrogen (1.8 ml/min) was utilized as carrier gas. The 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. The relative proportions of the EOs components were reported as percentages determined using peak-area normalization. The retention indices (RI) on HP-5 MS column, were identified using homologous series of (C8–C28) alkanes.


## 2.2.2. GC/MS Analysis

Chemical analysis was carried out using a Hewlett-Packard gas chromatograph (HP 6890) coupled with a mass spectrometer (HP 5973). The column utilized was HP-5MS (cross-linked 5% PHME siloxane; 30 m × 0.25 mm, film thickness 0.25µm). The column temperature was set at 50°C and increased to 250°C at a rate of 2°C/min. The 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. The ion source temperature of 230°C with ionization voltages of 70 eV and a scan mass range of 35–450 m/z. The identification of the components was also confirmed by comparing the elution order of the compounds with the relative retention indices reported in the literature.


## 2.3. Spider Mites

The adult spider mites ofT. 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). The 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.


## 2.4. Toxicity Test

The acaricidal tests were conducted according to the methodology of Sertkaya et al. (2010) with slight modifications. Firstly, various doses (39.06, 78.125, 156.25, 312.5, and 625µl/L) of each EO and the chemical acaricide (Kanemite, 15%) were prepared. Thereafter, 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. The toxicity was evaluated after 24 and 48 h of exposure, and the mortality was calculated using the formula of Abbott (1)(1)%Mortality Corrected=%Mortality Observed−%Mortality Control100−%Mortality Control×100.


## 2.5. Statistical Treatment

Mortality percentages were analyzed by the SPSS software (IBM SPSS Statistics 25.0). The obtained results were also examined with OriginPro 2021 software to determine the significant effect between mean values using the Tukey test (a probability ofp≤0.05 is regarded as statistically significant). The different principal component analysis was carried out by using the JMP Pro 14. The LD50 values were estimated using a probit regression analysis (with SPSS software) according to Finney’s mathematical methods [27].


## 3. Results

### 3.1. Chemical Composition of the Four Tested EOs

As presented in Table1, M. pulegium EO was dominated by r (+)-pulegone (74.03%), carvone (5.45%), and dihydrocarvone (3.66%). L. stoechas EO was characterized by camphor (43.97%), fenchone (30.39%), camphene (4.09%), borneol (2.92%), and α-pinene (2.84%); whereas 1,8 cineole (29.31%), camphor (24,66%), and α-pinene (12.76%) were the main components in R. officinalis. EO. Thymol (30.05%), carvacrol (25.33%), and γ-Terpinene (17.23%) presented the main volatile components in O. compactum EO.Table 1
The major chemical constituents of the four EOs.
Compound% Relative peak areaRIaRIbM. pulegiumL. stoechasR. officinalisO. compactumα-pinene9399320.422.8412.762.24Camphene953946—4.092.47Trα-Terpinene10021007——Tr3.45p-Cymene10261022—0.320.0911.221,8- cineole10331033—1.4231.15Trγ-Terpinene10621024—0.34—17.23Fenchone10881087—30.39—TrCamphor11431146—43.9726.960.14Borneol11651166—2.925.460.42Dihydrocarvone119411913.66———R (+)-pulegone1238123374.03—Tr—Carvone124212395.450.12—TrThymol12901293———30.05Carvacrol12981291———25.33Total %83.5686.4178.8990.08


### 3.2. Toxicity of EOs onT. urticae and E. orientalis

Figures1 and 2 illustrate the significant differences of the acaricidal effects of the four EOs and the chemical acaricide on T. urticae (Figure 1) and E. orientalis (Figure 2) according to mortality time (Figure 2(a)) and different concentration (Figure 2(b)). A significant difference (p<0.05) was noted between M. pulegium, R. officinalis, L. stoechas, and O. compactum (p=0.008) as well as between the four EOs and acaricide (p=0.009) in terms of mortality (%). In addition, a significant difference was observed between concentration (C1, C2, C3, and C4) of EOs and acaricide (p=0.004) in terms of mortality (%). However, no significant difference (p>0.05) was detected between EOs and acaricide (p=0.08) in terms of concentration C5, except R. officinalis EO for T. urticae. No significant difference was found between the mortality time of the four EOs and acaricide (p=0.18) in terms of T. urticae and E. orientalis mortality. It could be said that the mortality varies according to the oil and the acaricide tested and the treated mite species, generally; it was concentration-dependent. Indeed, M. pulegium and O. compactum EOs were the most effective against the two mites at all concentrations used, yet all EOs caused more than 90% of mortalities at the highest dose (C5: 625 µl/L). These results showed the important acaricidal potential of the different EOs. The chemical acaricide, Kanemite with the acequinocyl as the active molecule also displayed toxicity that varied in function of doses.Figure 1
Mortality rate (%) ofTetranychus urticae adults (a) according to mortality time. T1: 24 h; T2: 48 h, (b) according to various concentrations. C1: 39.06 µl/ml, C2: 78.125 µl/ml, C3: 156.25 µl/ml; C4: 312.5 µl/ml, C5: 625 µl/ml. Different letters indicate significant differences according to the Tukey test.
(a)(b)Figure 2
Mortality rate (%) ofEutetranychus orientalis adults (a) according to mortality time. T1: 24 h; T2: 48 h, (b) according to various concentrations. C1: 39.06 µl/ml, C2: 78.125 µl/ml, C3: 156.25 µl/ml; C4: 312.5 µl/ml, C5: 625 µl/ml. Different letters indicate significant differences according to the Tukey test.
(a)(b)The lethal doses (LD50) values obtained for each essential oil and the chemical acaricide are presented in Table 2. It was found that M. pulegium EO, O. compactum EO, and the chemical acaricide displayed the lowest LD50 values on adults of T. urticae after 24 h (65.78, 87.92, and 36.25 µl/L air, respectively) and after 48 h of treatment (56.1, 79.72, and 32.09 µl/L air, respectively). For E. orientalis, the lowest LD50 values were obtained for M. pulegium EO, R. officinalis EO, and the acaricide after 24 h (59.10, 88.2, and 34.07 µl/L air, respectively) and after 48 h of exposure (47.37, 82.44, and 29.15 µl/L air, respectively). The Chi-square test was not significant at 5% for all samples (EOs and the acaricide), which means goodness of fit.Table 2
LD50 values calculated for Tetranychus urticae and Eutetranychus orientalis.
Spider mites speciesSampleLD50 (µl/L air, 24 h, 95% CI∗)Slope (24 h)Intercept (24 h)dfX2LD50 (µl/L air, 48 h, 95% CI)dfX2Slope (48 h)Intercept (48 h)Tetranychidae urticaeM. pulegium65.78 (44.63–10110.12)1.69 ± 0.171.93 ± 0.3844.2156.1 (33.8–69.5)44.681.86 ± 0.181.75 ± 0.41L. stoechas100.03 (85.88–120.12)1.80 ± 0.241.41 ± 0.5542.6485.35 (65.15–115.12)42.891.87 ± 0.271.38 ± 0.60R. officinalis171.87 (163.24–184.96)1.59 ± 0.411.44 ± 0.9141.95141.51 (120.39–162.10)42.281.67 ± 0.481.40 ± 0.07O. compactum87.92 (68.15–99.65)1.29 ± 0.272.50 ± 0.5643.9279.72 (58.9–95.42)44.101.79 ± 0.331.27 ± 0.75Chemical acaricide36.25 (27.77–52.48)1.42 ± 0.372.79 ± 0.8344.6232.09 (19.81–61.10)44.91.72 ± 0.352.21 ± 0.78Eutetranychus orientalisM. pulegium59.10 (40.5–78.9)1.86 ± 0.151.71 ± 0.3543.9947.37 (39.80–63.65)44.511.91 ± 0.191.80 ± 0.42L. stoechas120.87 (101.19–146.62)1.79 ± 0.141.26 ± 0.3141.98100.35 (85.98–121.72)42.151.75 ± 0.151.50 ± 0.33R. officinalis88.2 (66.6–112.24)1.76 ± 0.281.57 ± 0.6343.1082.44 (70.06–109.2)43.841.89 ± 0.241.43 ± 0.54O. compactum112.77 (89.5–135.7)2.36 ± 0.240.16 ± 0.5442.9296.85 (81.45–140.16)43.82.38 ± 0.270.28 ± 0.61Chemical acaricide34.07 (25.10–50.23)1.49 ± 0.292.72 ± 0.6244.4929.15 (17.43–29.65)44.951.47 ± 0.272.84 ± 0.5795% CI: 95% confidence intervals; df: degree of freedom (Total);X2: Chi-square.


### 3.3. Toxicity of the EOs and the Acaricide onT. urticae and E. orientalis

As shown in Figure3, the four binary mixtures exhibited an important acaricidal effect on both T. urticae and E. orientalis after 24 and 48 h of treatment. A significant difference (p<0.05) was indicated in the mortality percentage of T. urticae between the M. pulegium EO + chemical acaricide (acequinocyl), L, stoechas EO + chemical acaricide (acequinocyl), and O. compactum EO + chemical acaricide (acequinocyl; p=0.014), while no significant difference was identified between M. pulegium EO + chemical acaricide (acequinocyl) and R. officinalis EO + chemical acaricide (acequinocyl; p=0.031). No significant difference (p>0.05) was identified between M. pulegium EO + chemical acaricide (acequinocyl), R. officinalis EO + chemical acaricide (acequinocyl), and O. compactum EO + chemical acaricide (acequinocyl; p=1.00), whereas a significant difference (p<0.05) was found between L. stoechas EO + chemical acaricide (acequinocyl) and the three mixtures in terms of the mortality percentage of E. orientalis (p=0.544). There was no significant in the mortality time of the four EOs and acaricide in terms of T. urticae and E. orientalis (p=0.115). The binary combination (M. pulegium EO + chemical acaricide) and (O. compactum EO + chemical acaricide) exerted a high acaricidal effect against T. urticae with 92 ± 2.74–99 ± 2.23% at 24 h and 95 ± 3.54–100 ± 0.00% of mortalities, respectively. However, the mixtures (M. pulegium EO + chemical acaricide, R. officinalis EO + chemical acaricide, and O. compactum EO + chemical acaricide) were the most effective on spider mites adults of E. orientalis with 99 ± 2.24% at 24 h and 100 ± 0.00% at 48 h of mortality. These results are very relevant and proved the acaricidal potential of EOs if they compared with the individual one.Figure 3
Mortality rate (%) ofTetranychus urticae adults according to various binary mixture of EOs and acaricide. (b) Mortality rate (%) of Eutetranychus orientalis adults according to various binary mixture of EOs and acaricide. Mixture 1: M. pulegium EO + acaricide; Mixture 2: L. stoechas EO + acaricide; Mixture 3: R. officinalis EO + acaricide; Mixture 4: O. compactum EO + acaricide. T1: 24 h; T2: 48 h. Different letters indicate significant differences according to the Tukey’s test (p≤0.05).
(a)(b)


### 3.4. Principal Component Analysis

#### 3.4.1. The Effect of the Main Compounds of the Four Tested EOs and Their Acaricidal Activity

The 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 againstE. orientalis and T. urticae in 24 and 48 h. The correlations of the different parameters studied are shown in a correlation matrix (Table 1).The loading plot (Figure4(a)) and the correlation matrix (Table 3) revealed some correlation between the variables studied. A strong positive correlation (>0.7) exists between (a) α-pinene and LD50 of T. urticae, (b) 1,8-cineole and LD50 of T. urticae, and (c) Borneol and LD50 of T. urticae. A medium positive correlation (0.6–0.5) exists between fenchone and LD50 of E. orientalis. A strong negative correlation (>−0.7) exists between (a) dihydrocarvone and LD50E. orientalis, (b) r(+)-pulegone and LD50E. orientalis, and (c) carvone and LD50 of E. orientalis. That means that when the amount of 1,8-cineole, α-pinene, and borneol compounds increased with increased LD50 of T. urticae, and when the LD50 of E. orientalis was low, the amount of dihydrocarvone, r(+)-pulegone, and carvone were important. The biplot (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. The M. pulegium was characterized by having the highest amount of r (+)-pulegone and carvone compounds and they require lowest concentration to eliminate E. orientalis.Figure 4
Principal component analyses of the main compounds in regards to the four essential oils. (a) Loading plot of the projection of the variables (main compounds) on the first and second principal components; (b) biplot of the projection of the variables on the individuals (the four essential oils) on the first and second principal components.
(a)(b)Table 3
Correlation matrix between the main compounds of the four essential oils and LD50 of E. orientalis and T. urticae.
α-pineneCampheneα-Terpinenep-Cymene1,8-Cineoleγ-TerpineneFenchoneCamphorBorneolDihydrocarvoneR(+)-pulegoneCarvoneThymolCarvacrolLD50E. orientalisLD50 T. urticaeα-pinene10.39−0.28−0.280.99−0.28−0.210.40.92−0.5−0.5−0.51−0.28−0.280.020.99Camphene0.391−0.55−0.530.32−0.530.8110.72−0.55−0.55−0.53−0.55−0.550.520.49α-Terpinene−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27p-Cymene−0.28−0.5311−0.361−0.31−0.53−0.46−0.35−0.35−0.36110.44−0.271,8-Cineole0.990.32−0.35−0.361−0.36−0.290.330.88−0.35−0.35−0.36−0.35−0.35−0.140.96γ-Terpinene−0.28−0.5311−0.361−0.32−0.53−0.47−0.34−0.34−0.35110.44−0.27Fenchone−0.210.81−0.33−0.31−0.29−0.3210.810.19−0.33−0.33−0.31−0.33−0.330.61−0.09Camphor0.41−0.54−0.530.33−0.530.8110.73−0.55−0.55−0.53−0.54−0.540.520.5Borneol0.920.72−0.47−0.460.88−0.470.190.731−0.58−0.58−0.58−0.47−0.470.210.95Dihydrocarvone−0.5−0.55−0.33−0.35−0.35−0.34−0.33−0.55−0.58111−0.33−0.33−0.87−0.59R(+)-pulegone−0.5−0.55−0.33−0.35−0.35−0.34−0.33−0.55−0.58111−0.33−0.33−0.87−0.59Carvone−0.51−0.53−0.34−0.36−0.36−0.35−0.31−0.53−0.58111−0.34−0.34−0.86−0.6Thymol−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27Carvacrol−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27LD50 E. orientalis0.020.520.420.44−0.140.440.610.520.21−0.87−0.87−0.860.420.4210.14LD50 T. urticae0.990.49−0.27−0.270.96−0.27−0.090.50.95−0.59−0.59−0.6−0.27−0.270.141Based on the result from Figures4(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. The effect of synergy could also be the origin of this activity.


#### 3.4.2. The Effect of the Concentrations of the Four Tested EOs and Their Acaricidal Activity

The second PCA permeated to identify the similar acaricidal activity effect of essential oil and concentration, the first principal component analysis was carried out. The individuals are represented by four essential oil samples (M. pulegium, L. stoechas, O. compactum, and R. officinalis) in five different concentrations (C1, C2, C3, C4, and C5). The variables are represented by acaricidal activity against E. orientalis and T. urticae in 24 and 48 h.The biplot (Figure5(a)) demonstrated that the distribution of individuals (concentration) in accordance with PC1 and PC2 shows that individuals were separated into three main groups. These results indicated that the concentration has an effect on the acaricidal activity, as previously shown by ANOVA. The first group consisted of C1 (39.06 µl/ml) and C2 (78.125 µl/ml), or the low concentration. The second group is composed of C4 (312.5 µl/ml) and C5 (625 µl/ml), or the high concentration. The third groups were C3 (156.25 µl/ml), or the medium concentration. This 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. The 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.Figure 5
Principal component analyses of acaricidal activity againstE. orientalis and T. urticae adults in regards to the four essential oils and concentrations. (a) Biplot of the projection of the variables and the individuals according to concentrations on the first and second principal components; (b) biplot of the projection of the variables and the individuals according to essential oils on the first and second principal components.
(a)(b)


#### 3.4.3. The Effect of the Four Mixtures Between Acaricide and Tested EOs and Their Acaricidal Activity

The 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).The biplot (Figure6) illustrated that the individuals (mixtures) were divided into three main groups. The first group was the mixture of M. pulegium EO + acaricide and R. officinalis EO + acaricide. The second group was the mixture of O. compactum EO + acaricide. The third group was L. stoechas EO + acaricide. The 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. This 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).Figure 6
Biplot of the projection of the acaricidal activity againstT. urticae and E. orientalis adults and the four mixtures on the first and second principal components.


### 3.5. The Hierarchical Cluster Analysis

The hierarchical cluster analysis (HCA) allowed us to better classify the studied samples (the four essential oils) according to the acaricidal activity against theE. 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.Figure 7
Hierarchical cluster analysis of the four mixtures againstE. orientalis and T. urticae adults.


## 3.1. Chemical Composition of the Four Tested EOs

As presented in Table1, M. pulegium EO was dominated by r (+)-pulegone (74.03%), carvone (5.45%), and dihydrocarvone (3.66%). L. stoechas EO was characterized by camphor (43.97%), fenchone (30.39%), camphene (4.09%), borneol (2.92%), and α-pinene (2.84%); whereas 1,8 cineole (29.31%), camphor (24,66%), and α-pinene (12.76%) were the main components in R. officinalis. EO. Thymol (30.05%), carvacrol (25.33%), and γ-Terpinene (17.23%) presented the main volatile components in O. compactum EO.Table 1
The major chemical constituents of the four EOs.
Compound% Relative peak areaRIaRIbM. pulegiumL. stoechasR. officinalisO. compactumα-pinene9399320.422.8412.762.24Camphene953946—4.092.47Trα-Terpinene10021007——Tr3.45p-Cymene10261022—0.320.0911.221,8- cineole10331033—1.4231.15Trγ-Terpinene10621024—0.34—17.23Fenchone10881087—30.39—TrCamphor11431146—43.9726.960.14Borneol11651166—2.925.460.42Dihydrocarvone119411913.66———R (+)-pulegone1238123374.03—Tr—Carvone124212395.450.12—TrThymol12901293———30.05Carvacrol12981291———25.33Total %83.5686.4178.8990.08


## 3.2. Toxicity of EOs onT. urticae and E. orientalis

Figures1 and 2 illustrate the significant differences of the acaricidal effects of the four EOs and the chemical acaricide on T. urticae (Figure 1) and E. orientalis (Figure 2) according to mortality time (Figure 2(a)) and different concentration (Figure 2(b)). A significant difference (p<0.05) was noted between M. pulegium, R. officinalis, L. stoechas, and O. compactum (p=0.008) as well as between the four EOs and acaricide (p=0.009) in terms of mortality (%). In addition, a significant difference was observed between concentration (C1, C2, C3, and C4) of EOs and acaricide (p=0.004) in terms of mortality (%). However, no significant difference (p>0.05) was detected between EOs and acaricide (p=0.08) in terms of concentration C5, except R. officinalis EO for T. urticae. No significant difference was found between the mortality time of the four EOs and acaricide (p=0.18) in terms of T. urticae and E. orientalis mortality. It could be said that the mortality varies according to the oil and the acaricide tested and the treated mite species, generally; it was concentration-dependent. Indeed, M. pulegium and O. compactum EOs were the most effective against the two mites at all concentrations used, yet all EOs caused more than 90% of mortalities at the highest dose (C5: 625 µl/L). These results showed the important acaricidal potential of the different EOs. The chemical acaricide, Kanemite with the acequinocyl as the active molecule also displayed toxicity that varied in function of doses.Figure 1
Mortality rate (%) ofTetranychus urticae adults (a) according to mortality time. T1: 24 h; T2: 48 h, (b) according to various concentrations. C1: 39.06 µl/ml, C2: 78.125 µl/ml, C3: 156.25 µl/ml; C4: 312.5 µl/ml, C5: 625 µl/ml. Different letters indicate significant differences according to the Tukey test.
(a)(b)Figure 2
Mortality rate (%) ofEutetranychus orientalis adults (a) according to mortality time. T1: 24 h; T2: 48 h, (b) according to various concentrations. C1: 39.06 µl/ml, C2: 78.125 µl/ml, C3: 156.25 µl/ml; C4: 312.5 µl/ml, C5: 625 µl/ml. Different letters indicate significant differences according to the Tukey test.
(a)(b)The lethal doses (LD50) values obtained for each essential oil and the chemical acaricide are presented in Table 2. It was found that M. pulegium EO, O. compactum EO, and the chemical acaricide displayed the lowest LD50 values on adults of T. urticae after 24 h (65.78, 87.92, and 36.25 µl/L air, respectively) and after 48 h of treatment (56.1, 79.72, and 32.09 µl/L air, respectively). For E. orientalis, the lowest LD50 values were obtained for M. pulegium EO, R. officinalis EO, and the acaricide after 24 h (59.10, 88.2, and 34.07 µl/L air, respectively) and after 48 h of exposure (47.37, 82.44, and 29.15 µl/L air, respectively). The Chi-square test was not significant at 5% for all samples (EOs and the acaricide), which means goodness of fit.Table 2
LD50 values calculated for Tetranychus urticae and Eutetranychus orientalis.
Spider mites speciesSampleLD50 (µl/L air, 24 h, 95% CI∗)Slope (24 h)Intercept (24 h)dfX2LD50 (µl/L air, 48 h, 95% CI)dfX2Slope (48 h)Intercept (48 h)Tetranychidae urticaeM. pulegium65.78 (44.63–10110.12)1.69 ± 0.171.93 ± 0.3844.2156.1 (33.8–69.5)44.681.86 ± 0.181.75 ± 0.41L. stoechas100.03 (85.88–120.12)1.80 ± 0.241.41 ± 0.5542.6485.35 (65.15–115.12)42.891.87 ± 0.271.38 ± 0.60R. officinalis171.87 (163.24–184.96)1.59 ± 0.411.44 ± 0.9141.95141.51 (120.39–162.10)42.281.67 ± 0.481.40 ± 0.07O. compactum87.92 (68.15–99.65)1.29 ± 0.272.50 ± 0.5643.9279.72 (58.9–95.42)44.101.79 ± 0.331.27 ± 0.75Chemical acaricide36.25 (27.77–52.48)1.42 ± 0.372.79 ± 0.8344.6232.09 (19.81–61.10)44.91.72 ± 0.352.21 ± 0.78Eutetranychus orientalisM. pulegium59.10 (40.5–78.9)1.86 ± 0.151.71 ± 0.3543.9947.37 (39.80–63.65)44.511.91 ± 0.191.80 ± 0.42L. stoechas120.87 (101.19–146.62)1.79 ± 0.141.26 ± 0.3141.98100.35 (85.98–121.72)42.151.75 ± 0.151.50 ± 0.33R. officinalis88.2 (66.6–112.24)1.76 ± 0.281.57 ± 0.6343.1082.44 (70.06–109.2)43.841.89 ± 0.241.43 ± 0.54O. compactum112.77 (89.5–135.7)2.36 ± 0.240.16 ± 0.5442.9296.85 (81.45–140.16)43.82.38 ± 0.270.28 ± 0.61Chemical acaricide34.07 (25.10–50.23)1.49 ± 0.292.72 ± 0.6244.4929.15 (17.43–29.65)44.951.47 ± 0.272.84 ± 0.5795% CI: 95% confidence intervals; df: degree of freedom (Total);X2: Chi-square.


## 3.3. Toxicity of the EOs and the Acaricide onT. urticae and E. orientalis

As shown in Figure3, the four binary mixtures exhibited an important acaricidal effect on both T. urticae and E. orientalis after 24 and 48 h of treatment. A significant difference (p<0.05) was indicated in the mortality percentage of T. urticae between the M. pulegium EO + chemical acaricide (acequinocyl), L, stoechas EO + chemical acaricide (acequinocyl), and O. compactum EO + chemical acaricide (acequinocyl; p=0.014), while no significant difference was identified between M. pulegium EO + chemical acaricide (acequinocyl) and R. officinalis EO + chemical acaricide (acequinocyl; p=0.031). No significant difference (p>0.05) was identified between M. pulegium EO + chemical acaricide (acequinocyl), R. officinalis EO + chemical acaricide (acequinocyl), and O. compactum EO + chemical acaricide (acequinocyl; p=1.00), whereas a significant difference (p<0.05) was found between L. stoechas EO + chemical acaricide (acequinocyl) and the three mixtures in terms of the mortality percentage of E. orientalis (p=0.544). There was no significant in the mortality time of the four EOs and acaricide in terms of T. urticae and E. orientalis (p=0.115). The binary combination (M. pulegium EO + chemical acaricide) and (O. compactum EO + chemical acaricide) exerted a high acaricidal effect against T. urticae with 92 ± 2.74–99 ± 2.23% at 24 h and 95 ± 3.54–100 ± 0.00% of mortalities, respectively. However, the mixtures (M. pulegium EO + chemical acaricide, R. officinalis EO + chemical acaricide, and O. compactum EO + chemical acaricide) were the most effective on spider mites adults of E. orientalis with 99 ± 2.24% at 24 h and 100 ± 0.00% at 48 h of mortality. These results are very relevant and proved the acaricidal potential of EOs if they compared with the individual one.Figure 3
Mortality rate (%) ofTetranychus urticae adults according to various binary mixture of EOs and acaricide. (b) Mortality rate (%) of Eutetranychus orientalis adults according to various binary mixture of EOs and acaricide. Mixture 1: M. pulegium EO + acaricide; Mixture 2: L. stoechas EO + acaricide; Mixture 3: R. officinalis EO + acaricide; Mixture 4: O. compactum EO + acaricide. T1: 24 h; T2: 48 h. Different letters indicate significant differences according to the Tukey’s test (p≤0.05).
(a)(b)


## 3.4. Principal Component Analysis

### 3.4.1. The Effect of the Main Compounds of the Four Tested EOs and Their Acaricidal Activity

The 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 againstE. orientalis and T. urticae in 24 and 48 h. The correlations of the different parameters studied are shown in a correlation matrix (Table 1).The loading plot (Figure4(a)) and the correlation matrix (Table 3) revealed some correlation between the variables studied. A strong positive correlation (>0.7) exists between (a) α-pinene and LD50 of T. urticae, (b) 1,8-cineole and LD50 of T. urticae, and (c) Borneol and LD50 of T. urticae. A medium positive correlation (0.6–0.5) exists between fenchone and LD50 of E. orientalis. A strong negative correlation (>−0.7) exists between (a) dihydrocarvone and LD50E. orientalis, (b) r(+)-pulegone and LD50E. orientalis, and (c) carvone and LD50 of E. orientalis. That means that when the amount of 1,8-cineole, α-pinene, and borneol compounds increased with increased LD50 of T. urticae, and when the LD50 of E. orientalis was low, the amount of dihydrocarvone, r(+)-pulegone, and carvone were important. The biplot (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. The M. pulegium was characterized by having the highest amount of r (+)-pulegone and carvone compounds and they require lowest concentration to eliminate E. orientalis.Figure 4
Principal component analyses of the main compounds in regards to the four essential oils. (a) Loading plot of the projection of the variables (main compounds) on the first and second principal components; (b) biplot of the projection of the variables on the individuals (the four essential oils) on the first and second principal components.
(a)(b)Table 3
Correlation matrix between the main compounds of the four essential oils and LD50 of E. orientalis and T. urticae.
α-pineneCampheneα-Terpinenep-Cymene1,8-Cineoleγ-TerpineneFenchoneCamphorBorneolDihydrocarvoneR(+)-pulegoneCarvoneThymolCarvacrolLD50E. orientalisLD50 T. urticaeα-pinene10.39−0.28−0.280.99−0.28−0.210.40.92−0.5−0.5−0.51−0.28−0.280.020.99Camphene0.391−0.55−0.530.32−0.530.8110.72−0.55−0.55−0.53−0.55−0.550.520.49α-Terpinene−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27p-Cymene−0.28−0.5311−0.361−0.31−0.53−0.46−0.35−0.35−0.36110.44−0.271,8-Cineole0.990.32−0.35−0.361−0.36−0.290.330.88−0.35−0.35−0.36−0.35−0.35−0.140.96γ-Terpinene−0.28−0.5311−0.361−0.32−0.53−0.47−0.34−0.34−0.35110.44−0.27Fenchone−0.210.81−0.33−0.31−0.29−0.3210.810.19−0.33−0.33−0.31−0.33−0.330.61−0.09Camphor0.41−0.54−0.530.33−0.530.8110.73−0.55−0.55−0.53−0.54−0.540.520.5Borneol0.920.72−0.47−0.460.88−0.470.190.731−0.58−0.58−0.58−0.47−0.470.210.95Dihydrocarvone−0.5−0.55−0.33−0.35−0.35−0.34−0.33−0.55−0.58111−0.33−0.33−0.87−0.59R(+)-pulegone−0.5−0.55−0.33−0.35−0.35−0.34−0.33−0.55−0.58111−0.33−0.33−0.87−0.59Carvone−0.51−0.53−0.34−0.36−0.36−0.35−0.31−0.53−0.58111−0.34−0.34−0.86−0.6Thymol−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27Carvacrol−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27LD50 E. orientalis0.020.520.420.44−0.140.440.610.520.21−0.87−0.87−0.860.420.4210.14LD50 T. urticae0.990.49−0.27−0.270.96−0.27−0.090.50.95−0.59−0.59−0.6−0.27−0.270.141Based on the result from Figures4(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. The effect of synergy could also be the origin of this activity.


### 3.4.2. The Effect of the Concentrations of the Four Tested EOs and Their Acaricidal Activity

The second PCA permeated to identify the similar acaricidal activity effect of essential oil and concentration, the first principal component analysis was carried out. The individuals are represented by four essential oil samples (M. pulegium, L. stoechas, O. compactum, and R. officinalis) in five different concentrations (C1, C2, C3, C4, and C5). The variables are represented by acaricidal activity against E. orientalis and T. urticae in 24 and 48 h.The biplot (Figure5(a)) demonstrated that the distribution of individuals (concentration) in accordance with PC1 and PC2 shows that individuals were separated into three main groups. These results indicated that the concentration has an effect on the acaricidal activity, as previously shown by ANOVA. The first group consisted of C1 (39.06 µl/ml) and C2 (78.125 µl/ml), or the low concentration. The second group is composed of C4 (312.5 µl/ml) and C5 (625 µl/ml), or the high concentration. The third groups were C3 (156.25 µl/ml), or the medium concentration. This 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. The 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.Figure 5
Principal component analyses of acaricidal activity againstE. orientalis and T. urticae adults in regards to the four essential oils and concentrations. (a) Biplot of the projection of the variables and the individuals according to concentrations on the first and second principal components; (b) biplot of the projection of the variables and the individuals according to essential oils on the first and second principal components.
(a)(b)


### 3.4.3. The Effect of the Four Mixtures Between Acaricide and Tested EOs and Their Acaricidal Activity

The 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).The biplot (Figure6) illustrated that the individuals (mixtures) were divided into three main groups. The first group was the mixture of M. pulegium EO + acaricide and R. officinalis EO + acaricide. The second group was the mixture of O. compactum EO + acaricide. The third group was L. stoechas EO + acaricide. The 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. This 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).Figure 6
Biplot of the projection of the acaricidal activity againstT. urticae and E. orientalis adults and the four mixtures on the first and second principal components.


## 3.4.1. The Effect of the Main Compounds of the Four Tested EOs and Their Acaricidal Activity

The 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 againstE. orientalis and T. urticae in 24 and 48 h. The correlations of the different parameters studied are shown in a correlation matrix (Table 1).The loading plot (Figure4(a)) and the correlation matrix (Table 3) revealed some correlation between the variables studied. A strong positive correlation (>0.7) exists between (a) α-pinene and LD50 of T. urticae, (b) 1,8-cineole and LD50 of T. urticae, and (c) Borneol and LD50 of T. urticae. A medium positive correlation (0.6–0.5) exists between fenchone and LD50 of E. orientalis. A strong negative correlation (>−0.7) exists between (a) dihydrocarvone and LD50E. orientalis, (b) r(+)-pulegone and LD50E. orientalis, and (c) carvone and LD50 of E. orientalis. That means that when the amount of 1,8-cineole, α-pinene, and borneol compounds increased with increased LD50 of T. urticae, and when the LD50 of E. orientalis was low, the amount of dihydrocarvone, r(+)-pulegone, and carvone were important. The biplot (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. The M. pulegium was characterized by having the highest amount of r (+)-pulegone and carvone compounds and they require lowest concentration to eliminate E. orientalis.Figure 4
Principal component analyses of the main compounds in regards to the four essential oils. (a) Loading plot of the projection of the variables (main compounds) on the first and second principal components; (b) biplot of the projection of the variables on the individuals (the four essential oils) on the first and second principal components.
(a)(b)Table 3
Correlation matrix between the main compounds of the four essential oils and LD50 of E. orientalis and T. urticae.
α-pineneCampheneα-Terpinenep-Cymene1,8-Cineoleγ-TerpineneFenchoneCamphorBorneolDihydrocarvoneR(+)-pulegoneCarvoneThymolCarvacrolLD50E. orientalisLD50 T. urticaeα-pinene10.39−0.28−0.280.99−0.28−0.210.40.92−0.5−0.5−0.51−0.28−0.280.020.99Camphene0.391−0.55−0.530.32−0.530.8110.72−0.55−0.55−0.53−0.55−0.550.520.49α-Terpinene−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27p-Cymene−0.28−0.5311−0.361−0.31−0.53−0.46−0.35−0.35−0.36110.44−0.271,8-Cineole0.990.32−0.35−0.361−0.36−0.290.330.88−0.35−0.35−0.36−0.35−0.35−0.140.96γ-Terpinene−0.28−0.5311−0.361−0.32−0.53−0.47−0.34−0.34−0.35110.44−0.27Fenchone−0.210.81−0.33−0.31−0.29−0.3210.810.19−0.33−0.33−0.31−0.33−0.330.61−0.09Camphor0.41−0.54−0.530.33−0.530.8110.73−0.55−0.55−0.53−0.54−0.540.520.5Borneol0.920.72−0.47−0.460.88−0.470.190.731−0.58−0.58−0.58−0.47−0.470.210.95Dihydrocarvone−0.5−0.55−0.33−0.35−0.35−0.34−0.33−0.55−0.58111−0.33−0.33−0.87−0.59R(+)-pulegone−0.5−0.55−0.33−0.35−0.35−0.34−0.33−0.55−0.58111−0.33−0.33−0.87−0.59Carvone−0.51−0.53−0.34−0.36−0.36−0.35−0.31−0.53−0.58111−0.34−0.34−0.86−0.6Thymol−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27Carvacrol−0.28−0.5511−0.351−0.33−0.54−0.47−0.33−0.33−0.34110.42−0.27LD50 E. orientalis0.020.520.420.44−0.140.440.610.520.21−0.87−0.87−0.860.420.4210.14LD50 T. urticae0.990.49−0.27−0.270.96−0.27−0.090.50.95−0.59−0.59−0.6−0.27−0.270.141Based on the result from Figures4(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. The effect of synergy could also be the origin of this activity.


## 3.4.2. The Effect of the Concentrations of the Four Tested EOs and Their Acaricidal Activity

The second PCA permeated to identify the similar acaricidal activity effect of essential oil and concentration, the first principal component analysis was carried out. The individuals are represented by four essential oil samples (M. pulegium, L. stoechas, O. compactum, and R. officinalis) in five different concentrations (C1, C2, C3, C4, and C5). The variables are represented by acaricidal activity against E. orientalis and T. urticae in 24 and 48 h.The biplot (Figure5(a)) demonstrated that the distribution of individuals (concentration) in accordance with PC1 and PC2 shows that individuals were separated into three main groups. These results indicated that the concentration has an effect on the acaricidal activity, as previously shown by ANOVA. The first group consisted of C1 (39.06 µl/ml) and C2 (78.125 µl/ml), or the low concentration. The second group is composed of C4 (312.5 µl/ml) and C5 (625 µl/ml), or the high concentration. The third groups were C3 (156.25 µl/ml), or the medium concentration. This 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. The 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.Figure 5
Principal component analyses of acaricidal activity againstE. orientalis and T. urticae adults in regards to the four essential oils and concentrations. (a) Biplot of the projection of the variables and the individuals according to concentrations on the first and second principal components; (b) biplot of the projection of the variables and the individuals according to essential oils on the first and second principal components.
(a)(b)


## 3.4.3. The Effect of the Four Mixtures Between Acaricide and Tested EOs and Their Acaricidal Activity

The 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).The biplot (Figure6) illustrated that the individuals (mixtures) were divided into three main groups. The first group was the mixture of M. pulegium EO + acaricide and R. officinalis EO + acaricide. The second group was the mixture of O. compactum EO + acaricide. The third group was L. stoechas EO + acaricide. The 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. This 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).Figure 6
Biplot of the projection of the acaricidal activity againstT. urticae and E. orientalis adults and the four mixtures on the first and second principal components.


## 3.5. The Hierarchical Cluster Analysis

The hierarchical cluster analysis (HCA) allowed us to better classify the studied samples (the four essential oils) according to the acaricidal activity against theE. 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.Figure 7
Hierarchical cluster analysis of the four mixtures againstE. orientalis and T. urticae adults.


## 4. 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]. The 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], The 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]. The 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].Thymol, a hydrophobic compound, constitutes the major monoterpene inO. compactum; it can penetrate the compartment membrane, which diminishes its impermeability, this mechanism stimulates the entry of other components into the cytoplasm [35, 36]. There is also evidence in the literature that thymol and eugenol have a neurotoxic effect on arthropods, with different mechanisms of action. Thymol 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]. This 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]. The essential oil of M. pulegium was very toxic against the strawberry spider mite Tetranychus turkestani Ugarov and Nikolskii, 1937 (Acari: Tetranychidae) [19]. The same results in another study were observed on T. urticae, a serious pest of many agricultural crops. The phytotoxicity of M. pulegium was significant against this pest after 24 h [40, 41].The 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 LD50, dihydrocarvone and carvone. Thus, 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. The results show that most of the monoterpenes with high concentrations have significantly insecticidal and acaricidal effects on the tested pests [43, 44].The second PCA showed thatM. 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 LC50 and LC90 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).The third PCA and HCA illustrated that the activity ofO. compactum EO has been ameliorated when it is mixed with the acaricide (synergetic effect). The 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. The search for synergistic mixtures aims to reduce the concentrations needed by increasing the biological activity against the target organism. This reduction of concentration may also result in decreased toxicological and environmental risks, and lower production cost [48, 49].


## 5. Conclusions

This scientific investigation highlighted the acaricidal effect of four essential oils against two important spider mites involved in agriculture damage. The 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%. These plant-based natural products can be applied in large field applications using low concentrations (based on the LD50 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. Their 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.



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*Source: 2004623-2022-08-22.xml*