Botanical extracts, including essential oils, are promising alternatives to synthetic insecticides for pest control. In this study, we evaluated the fumigant toxicity of an essential oil extracted from Thymus capitatus (L.) Hoffmanns. & Link against the citrus mealybug, Planococcus citri Risso (Hemiptera: Pseudococcidae), and its coccinellid predator Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae) under laboratory conditions. Gas chromatography–mass spectrometry analysis indicated that the major chemical compounds identified from T. capitatus were carvacrol (65.15%), followed by p-cymene (11.79%) and γterpinene (7.48%). High mortality levels were registered for P. citri larvae (up to 100%) and adults (up to 96%) when exposed to the tested essential oil. The median lethal concentration values calculated for P. citri adults were higher than for larvae. Thymus capitatus essential oil applied at 10 and 20 µL/Lair showed high toxicity towards C. montrouzieri adults. These results highlighted the efficacy of T. capitatus essential oil as a promising tool to control P. citri in Tunisia. However, the adverse effects of this oil towards C. montrouzieri should be taken into consideration to enhance its practical implication in integrated pest management.
The citrus mealybug, Planococcus citri Risso (Hemiptera: Pseudococcidae), is a major pest of a variety of host plants due to its direct feeding on the plant and as a vector of plant pathogens (Blaisdell et al. 2020). This pest is known as one of the most serious polyphagous pests throughout the Mediterranean basin attacking plants belonging to 70 families (Franco et al. 2009, Mishra et al. 2021). It thrives under the mild and tropical climate of the region (Goldasteh et al. 2009, Uygun and Satar 2008, Yayla et al. 2020), mainly in citrus orchards and nurseries (Bodenheimer 1951, Rao et al. 2006, Rung et al. 2009). Planococcus citri has a strong negative impact on yields by reducing plant vigor (Morandi Filho et al. 2015, Zappalà 2010), which may lead to lowering fruit quality and marketable value (Jaouad et al. 2020). High economic losses have been reported, with defoliation and fruit drop reaching 80% and 100%, respectively, in citrus crops (Zappalà 2010). Management of P. citri has largely depended on the use of a few selective insecticides, including methidathion (Brück et al. 2009, Mansour et al. 2010), malathion (Jacas 2010, Kerns et al. 2001), spirotetramat, and buprofezin (Satar et al. 2013). However, problems of resistance development to some active ingredients have already been reported worldwide (Mansour et al. 2010, Venkatesan et al. 2016). Implementation of effective and sustainable control tools by application of biopesticides or release of natural enemies are recommended as alternatives for pest control (Afifi et al. 2010, Kairo et al. 2013, Pérez-Rodríguez et al. 2019, Raveau et al. 2020, Regnault-Roger et al. 2012, Saljoqi et al. 2015). Essential oils are considered as a promising tool for pest management thanks to their properties as fumigants, antifeedants, contact insecticides, and repellents (Raveau et al. 2020). These natural compounds may also influence the growth rate, behavior, and reproduction of insect pests (Isman et al. 2008, Wang et al. 2006).
In this context, the aim of this work was to explore the chemical composition of essential oil extracted from Thymus capitatus (L.) Hoffmanns. & Link collected from the north of Tunisia. We further aimed to evaluate its fumigant toxicity against P. citri and a coccinellid predator, Cryptolaemus montrouzieri Mulsant (Coleoptera: Coccinellidae), under laboratory conditions.
Materials and Methods
Insects.Planococcus citri eggs, larvae and adults were collected from infested citrus, Citrus sinensis (L.) Osbeck var. ‘Thomson’ located in Mornag (northeastern Tunisia; N 36°40′54″, E 0°15′30″) and transported to the laboratory. The pest was reared for several generations on sugar pie pumpkin, Cucurbita pepo Grebenshchikiv, (weighing 2–4 kg) placed in a cage (50 × 50 × 50 cm) under controlled climatic conditions (28 ± 2°C; 65 ± 5% relative humidity [RH]; 12:12 h light:dark [L:D]). New pumpkins were added whenever necessary to avoid possible contamination by mites or fungi.
Cryptolaemus montrouzieri Mulsant beetles (72 h old) were purchased from Nutriplant (Tunisia) and reared for several generations on P. citri population under laboratory conditions (25 ± 2°C; 65 ± 5% RH; 16:18 h L:D).
Essential oil extraction and chemical identification.Thymus capitatus aerial parts (flower, leaves, and stems) were collected from the north of Tunisia (Governorate of Zaghouan, N 36°20′25″, E 10°03′46″) during June 2019. The essential oil was extracted by steam distillation of 1 kg of fresh aerial parts for 3 h using a Clevenger-type apparatus. The extracted essential oil was stored at 4°C.
The essential oil was analyzed and quantified by gas chromatography–mass spectrometry (GC-MS) and fast GC. GC-MS analyses were conducted on a thermo trace MS Finnigan mass-selective detector equipped with an Optima 5 MS (Macherey-Nagel) capillary column (30 m × 0.25 mm internal diameter [I.D.], 0.25-µm film thickness) and a split/splitless injector (splitless mode) at 250°C. The oven temperature was programmed from 40 to 210°C. Helium was the carrier gas at 1 ml/min. Volatile compounds were identified by comparing the obtained mass spectra with those from the Wiley 275-L spectral library and with their retention indices. Retention indices were determined relative to the retention times of a series of nalkane standards (C9-C30, Sigma-Aldrich, 0.025 µg/µl in n-hexane), measured under the chromatographic conditions described above, and compared with literature values (Adams 2001).
Fast GC analyses were conducted on a Thermo Ultra Fast Trace GC gas chromatograph operated with a split/splitless injector and a Thermo AS 3000 autosampler (Thermo Electron Corp.). The GC system was equipped with an ultrafast module (UFM) incorporating a direct resistively heated column (Thermo Electron Corp.): UFC-5, 5% phenyl, 5 m × 0.1 mm I.D., 0.1-µm film thickness. The following chromatographic conditions were used for obtaining suitable peak resolution. The UFM temperature program was as follows: initial temperature at 40°C, held for 0.1 min, ramp 1 at 30°C/min to 95°C, ramp 2 at 35°C/min to 155°C, ramp 3 at 200°C/min to 280°C, held for 0.5 min. Injection temperature was 240°C; injection volume, 1 µl; carrier gas was helium, at constant flow rate of 0.5 ml/min; and split ratio, 1:100. The GC unit has a high-frequency fast flame ionization detector (300 Hz), at 250°C. Hydrogen gas flow was 35 ml/min; air flow, 350 ml/min; makeup gas flow (N2), 30 ml/min. Data processing was by Chromcard software (version 2.3.3).
Toxicity bioassays. The insecticidal activity of T. capitatus essential oil was determined by fumigation method using Whatman filter papers impregnated with various concentrations of the essential oil. Planococcus citri or C. montrouzieri specimens were transferred from citrus leaves maintained on watered cotton and placed individually in 1-L plastic bottles using a soft brush. Five concentrations (1, 5, 7, 10, and 20 µl/Lair) were tested for P. citri. However, only two high concentrations (10 and 20 µl/Lair) were tested for C. montrouzieri. Tap water was used as the untreated control. Five replications were conducted for each concentration as well for the untreated control for both trials. For the P. citri trial, 20 larvae (L1) and 20 adults were used separately for each concentration. However, only 20 C. montrouzieri adults were used in that trial for both concentrations. Bottles serving as the bioassay arenas were maintained in a controlled climatic room (28 ± 2°C, 65 ± 5% RH, and 16:8 h L:D). Mortality was recorded after 4, 10, 24, 48, and 72 h of treatment. Insects were considered dead when neither leg nor antennae showed movement when probed with a fine hair brush while being observed under a binocular microscope (Leica® model MS5).
Statistical analysis. SPSS statistical software, version 21.0, was used to perform all statistical analysis. Data, already checked for their homogeneity (Levene test) and normality (Shapiro-Wilk test), were subjected to repeated measures analysis followed by one-way analysis of variance to assess the effect of T. capitatus essential oil on insect survival. Means were separated using the Tukey HSD test at P = 0.05. A probit test was performed to calculate the concentration-mortality responses (Finney et al. 1971).
Characterization of the essential oil. The volatile compounds in the T. capitatus essential oil obtained by hydrodistillation are shown in Table 1. The yield of the extracted oil from the aerial part was 0.93%. Ten representative components were identified by GC-MS and GC–flame-ionization detection, representing 94.76% of the total essential oil constituents. Carvacrol was the most abundant compound (65.15%), followed by p-cymene (11.79%), γ-terpinene (7.48%), E-caryophyllene (4.44%), myrcene (2.19 %), and linalool (1.32%).
Insecticidal activity against P. citri larvae and adults. Obtained data fit the linear model and indicated the suitability of the model to estimate the median lethal concentration (LC50) of the essential oil against P. citri (Table 2). The LC50 calculated for P. citri adults was higher than for larvae (Table 2). Based on these results, high levels of T. capitatus essential oil were required to kill 50% of exposed larvae and adults after 4 and 10 h of treatment (Table 2).
Fumigant toxicity. The T. capitatus essential oil was toxic towards P. citri larvae and adults. The highest levels of mortality were obtained at highest concentrations (10 and 20 µl/Lair) tested for both larvae and adults during the entire study period. Our results indicate that larval and adult mortality numbers depended on oil concentration.
Repeated-measures analysis indicated that the application rate (F = 21.94; df = 1; P < 0.0001) and the insect stage (F = 4.79; df = 1; P = 0.03) had a significant effect on the mortality obtained at the five time intervals (4, 10, 24, 48, and 72 h) after treatment. There was a significant difference between the control and all tested rates for larvae (F = 10.52; df = 5, 149; P < 0.0001) (Table 3) and adults (F = 24.42; df = 5, 149; P < 0.0001) (Table 4). Moreover, statistical analysis indicated that the mortality was influenced by the insect stage (F = 5.16; df = 1, 299; P = 0.02).
Response of C. montrouzieri adults to essential oil. Thymus capitatus essential oil showed high toxicity against C. montrouzieri adults at the two tested concentrations (10 and 20 µl/Lair). Three days after treatment, the oil applied at 20 µl/Lair killed more than 90% of exposed beetles.
Repeated-measures analysis indicated that the applied concentrations (F = 9.51; df = 1; P < 0.001) had a significant effect on the mortality at the five time intervals (4, 10, 24, 48, and 72 h) after treatment. There was a significant difference between the control and the two concentrations in mortality response at 10 µl/Lair (F = 14.76; df = 1, 49; P < 0.0001) and at 20 µl/Lair (F = 17.22; df = 1, 49; P < 0.0001).
In this study, 10 compounds were identified from the essential oil extracted from T. capitatus. Yield of the extracted essential oil was 0.93%. Previous studies reported that the yield of T. capitatus essential oil in Tunisia may differ according the regions from which plants grew (Akrout et al. 2010, Ben Ghnaya-Chakroun et al. 2015, Bounatirou et al. 2007, Mkaddem et al. 2010, Tammar et al. 2018, Zaïri et al. 2019). In the north of Tunisia, the yields of T. capitatus essential oil collected from three regions—Jendouba and Ain Tounine (Governorate of Jandouba) and Haouaria (Governorate of Nabeul)—were 3.4%, 2.6%, and 2.8%, respectively (Bounatirou et al. 2007). Also, Ben Ghnaya-Chakroun et al. (2015) reported that T. capitatus yield was 1.46% in Wed Ezzarga (Governorate of Beja). Recently, Tammar et al. (2018) indicated that the yield of T. capitaus obtained from the governorates of Bizerte, Kef, Nabeul, and Ben arous was 2.37 ± 0.18%, 1.64 ± 0.18%, 1.39 ± 0.19%, and 1.39 ± 0.11%, respectively. In the south of Tunisia, previous studies indicated that the yield of essential oil from T. capitatus leaves from Matmata (Governorate of Gabès) was 1.2% (Mkaddem et al. 2010) and 2.6% from Beni-Khedache (Governorate of Medenine) (Akrout et al. 2010). Zaïri et al. (2019) showed that T. capitatus yield was 0.82% in the Governorate of Kairouan in the center of Tunisia. The yield of T. capitatus essential oil varied among countries. For example, in Libya, El-Jalel et al. (2018) showed the yield of this oil varies depending on the latitude of the region. They demonstrated that the yield of the oil was higher at lower elevations (1.5%) compared to higher ones (1.06%). In Algeria, Goudjil et al. (2020) reported that the yield of T. capitatus essential oil was 1.56%. In Egypt, the yield of T. capitatus extracted plant oil was 0.5% (Salama et al. 2012).
Here, carvacrol was the major component (65.15%) identified from the extract. In fact, in Tunisia and regardless of the region, T. capitatus essential oil was characterized by a predominance of phenols due to the high amount of carvacrol (Akrout et al. 2010, Ben Ghnaya et al. 2015, Bounatirou et al. 2007, Mkaddem 2010, Moujahed et al. 2011, Tammar et al. 2018, Zaïri et al. 2019). In Tunisia, according to Hosni et al. (2013), the chemical composition of T. capitatus collected from Mograne (Governorate of Zaghouan) was carvacrol (58.66–81.49%; major component), p-cymene (3.83–13.17%), and γ-terpinene (7.81–3.16%). However, Mkaddem et al. (2010) showed that thymol was the major component of T. capitatus essential oil collected from Matmata (Governorate of Gabes), with 89.06%, followed by p-cimene with 5.04%, and γ-terpinene with 3.19%. In Algeria (Tiaret, western Algeria), thymol was the major component with 51.22%, followed by carvacrol with 12.59% and γ-terpinene with 10.3% (Goudjil et al. 2020). In Libya, Giweli et al. (2016) demonstrated that the most abundant composition of T. capitatus essential oil was carvacrol (68.19%), followed by thymol (12.29%) and γ-terpinene (3.09%). In Morrocco, Aissaoui et al. (2018) showed that T. capitatus is composed mostly of carvacrol (55.59%), followed by p-cymene (11.23%), and α-pinene (0.56%). Variation in the chemical composition of essential oils could be related to various factors including the environmental conditions (e.g., climatic conditions) and the time of harvest (Fidan et al. 2019, Msaada et al. 2012). The fluctuation in carvacrol percentage may be due to many bioclimatic factors such as the elevation, longitude, and latitude (Mkaddem 2010, Tammar et al. 2018) as well the origin of plant (Santiago et al. 2014).
The present study determined the insecticidal activity of the T. capitatus essential oil against P. citri and C. montrouzieri. Mortality of both stages of the two insect species were directly related to concentration of the oil. Several studies report the toxicity of Thymus genus against hemipteran pests worldwide. In this context, Khaled et al. (2017) demonstrated that T. capitatus essential oil showed a fumigant toxicity against Myzus persicae Sulzer (Hemiptera: Aphididae), with an LC50 of about 20.01 µl/Lair, after 24 h of treatment. Al-Mazra'awi and Ateyyat (2009) demonstrated that T. capitatus essential oil was repellent towards Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) adults. According to Attia et al. (2012) these variations in toxicity between different species of plants could be explained by the proportion of each component present in each essential oil. The genus Thymus is frequently applied on P. citri. For example, Erdemir and Erler (2017) reported that Thymus vulgaris (L.) essential oil had a repellent activity against P. citri adults with 88.8% at a concentration of 5 ml/L water after 96 h following application.
Our data clearly demonstrated high toxicity of T. capitatus essential oil against C. montrouzieri adults. High levels of mortality occurred with the two applied concentrations (10 and 20 µl/Lair). Recently, Bakkali-Aissaoui and Elamrani (2020) demonstrated that T. capitatus essential oil was less toxic to adults of Phytoseiulus persimilis Athias-Henroit (Acari: Phytoseiidae) at a concentration of 1% with 16.82% mortality. Santiago et al. (2014) showed that extracts of Chenopodium ambrosioides L. exhibited an insecticidal effect on C. montrouzieri adults after 144 h of exposure with a LC50 of 1.4 × 10 3. Bibi et al. (2021) indicated that the citrus oil was safe to C. montrouzieri adults with low mortality of 15–25% after 24 h of exposure, reporting a LC50 of 0.10 µl/ml. Fand et al. (2012) reported that aqueous garlic extract applied at concentrations of 0.5%, 1.0%, and 1.5% was also safe with less than 20% mortality to second-instar C. montrouzieri after 24, 48, and 72 h of treatment.
In conclusion, the biological activity of T. capitatus is likely related to its chemical composition. Our data showed promising insecticidal potential of T. capitatus essential with lower LC50 values against P. citri larvae and adults under laboratory conditions; this oil also caused high mortality levels of adults of the predatory beetle C. montrouzieri. Further studies should be conducted to confirm the efficacy of extracted T. capitatus essential oil under field conditions. Possible integration into integrated pest management programs should be considered.
This work was supported by the National Agronomic Institute of Tunisia. Rafika Alloui-Griza benefited from a doctoral fellowship as a part of Algerian–Tunisian cooperation from the Ministry of Higher Education and Scientific Research, Algeria.