Preliminary Study on Insecticidal Potential and Chemical Composition of Five Rutaceae Essential Oils against Thrips flavus (Thysanoptera: Thripidae)

To meet the demand for novel pest management strategies to combat the development of insecticide resistance, plant essential oils may be a promising alternative source. This study investigated the insecticidal activity of five essential oils from the Rutaceae plant family against Thrips flavus Schrank (Thysanoptera: Thripidae) under laboratory conditions. The plant essential oils were citrus oil (Citrus reticulata Blanco), Chuan-shan pepper oil (Zanthoxylum piasezkii Maxim.), zanthoxylum oil (Zanthoxylum bungeanum Maxim.), pomelo peel oil (Citrus maxima (Burm.) Merr.) and orange leaf oil (Citrus sinensis (L.) Osbeck). Among the essential oils evaluated, orange leaf oil (LC50 = 0.26 g/L), zanthoxylum oil (LC50 = 0.27 g/L), and pomelo peel oil (LC50 = 0.44 g/L) resulted in a higher gastric toxicity under laboratory conditions. The results of the pot experiment also showed that orange leaf oil (93.06 ± 3.67% at 540.00 g a.i.·hm−2, 97.22 ± 1.39% at 720 g a.i.·hm−2, 100.00% at 900.00 g a.i.·hm−2) zanthoxylum oil (98.73 ± 1.27% at 900 g a.i.·hm−2), and pomelo peel oil (100.00% at 900 g a.i.·hm−2) exhibited a higher control efficacy, being the most effective against T. flavus after 7 days of treatment. The essential oil components were then identified by gas chromatography–mass spectrometry (GC–MS). The insecticidal activity of orange leaf oil, pomelo peel oil, and zanthoxylum oil could be attributed to their main constituents, such as methyl jasmonate (50.92%), D-limonene (76.96%), and linalool (52.32%), respectively. In the olfactory test, adult T. flavus were attracted by zanthoxylum oil and Chuan-shan pepper oil. We speculated that linalool might be the key signaling compound that attracts T. flavus. These results showed that orange leaf oil, zanthoxylum oil, and pomelo peel oil exhibited insecticidal activities under controlled conditions. They can be implemented as effective and low-toxicity botanical insecticides and synergistic agents against T. flavus.


Introduction
Eurasian yellow flower thrips, Thrips flavus Schrank (Thysanoptera: Thripidae), is a worldwide phytophagous pest distributed in Asia and Europe and recorded from a wide range of host plants [1][2][3]. T. flavus is a host to a strain of the tomato spotted wilt virus (TSWV-W) on watermelon (Citrullus lanatus (Thunb.) Matsum. et Nakai) in India [4,5], which is highly invasive to other regions since it has well-developed dispersion mechanisms [6]. T. flavus has been increasingly recognized as a dominant pest of Compositae (Asteraceae), Leguminosae (Fabaceae), and other plant families during the flowering stage in north China [7]. This pest generally results in curling, deformation, and withering of the leaves and early senescence or deformation of the flowers [8]. Currently, the field management of T. flavus depends on chemical insecticides [9,10]. However, long-term insecticide use would lead to the rapid development of insecticide resistance in pest thrips [11,12].

Laboratory Bioassay
In the gastric toxicity test, the orange leaf oil had the highest biological activity, with an LC 50 of 0.26 g/L, followed by zanthoxylum oil, with an LC 50 of 0.27 g/L, and pomelo peel oil, with an LC 50 of 0.44 g/L. Citrus oil was the least active, with an LC 50 of  (Table 2). This indicated that among the Rutaceae EOs tested, the orange leaf oil, zanthoxylum oil, and pomelo peel oil possessed a higher gastric toxicity. The control efficacy of citrus oil against T. flavus increased with increasing concentration and treatment time (Table 3). Among them, except for the concentrations of 900.00 and 3600.00 g a.i.·hm −2 , from day 1 to day 7, the control efficacy initially showed an increasing trend, which then decreased. Notably, the differences between the concentrations evaluated diminished as the treatment time increased from 1 day to 7 days ( Table 3). The control efficacy of 900 g a.i.·hm −2 was significantly lower than that of 2700.00, 3600.00, and 4500.00 g a.i.·hm −2 after 1 day (F = 9.3950, df = 4, p = 0.002). No significant differences were observed between the five concentrations after 3 days (F = 0.3070, df = 4, p = 0.8666) and after 7 days (F = 0.3960, df = 4, p = 0.8069). The highest control efficacy of citrus oil was 62.03 ± 4.38% at 4500.00 g a.i.·hm −2 after 7 days. Note: Significant differences (p < 0.05) are indicated by different lowercase letters following the data in the same column for the same essential oil. The same as below.

Control Efficacy of Pomelo Peel Oil against T. flavus
The control efficacy of pomelo peel oil against T. flavus increased with the increasing concentration and treatment time ( Table 3). The control efficacy of 900.00 g a.i.·hm −2 was the highest compared to the other applied concentrations after 1 day (F = 26.1330, df = 4, p = 0.0001). The control efficacy of 900.00 g a.i.·hm −2 was significantly higher than that of 180.00 and 360.00 g a.i.·hm −2 after 3 days (F = 17.8250, df = 4, p = 0.0002) and reached its highest value after 7 days with 100.00% efficacy (F = 37.2250, df = 4, p = 0.0001), which was the highest control efficacy of pomelo peel oil measured overall.

Control Efficacy of Orange Leaf Oil against T. flavus
The control efficacy of orange leaf oil against T. flavus increased with the increasing concentration and treatment time ( Table 3). The control efficacy of 720.00 and 900.00 g a.i.· hm −2 was significantly higher than the other concentrations after 1 day (F = 11.601, df = 4, p = 0.0009). The control efficacy of 900.00 g a.i.·hm −2 was significantly higher than the other concentrations evaluated after 3 days (F = 18.9550, df = 4, p = 0.0001). The control efficacy of 720.00 and 900.00 g a.i.·hm −2 was significantly higher than that of 180.00 and 360.00 g a.i.·hm −2 after 7 days (F = 11.4030, df = 4, p = 0.0001). The highest control efficacy of orange leaf oil was 100.00% at 900.00 g a.i.·hm −2 after 7 days.

Discussion
In agricultural pest control, pesticide resistance is becoming increasingly prominent; thus, a higher demand exists for developing and applying efficient and safe insecticides [5]. Plant essential oils can be used in pest thrips management strategies and reduce the overuse of synthetic pesticides [44]. The present study was conducted to evaluate the insecticidal activity of five Rutaceae EOs for T. flavus control, followed by a preliminary identification of their chemical composition. Among the EOs tested, orange leaf oil (LC50 = 0.26 g/L), zanthoxylum oil (LC50 = 0.27 g/L), and pomelo peel oil (LC50 = 0.44 g/L) showed the highest gastric toxicity. The pot experiments' results demonstrated that orange leaf oil (93.06 ± 3.67% at 540.00 g a.i.·hm −2 , 97.22 ± 1.39% at 720 g a.i.·hm −2 , 100.00% at 900.00 g a.i.·hm −2 ), zanthoxylum oil (98.73 ± 1.27% at 900 g a.i.·hm −2 ), and pomelo peel oil (100.00% at 900 g a.i.·hm −2 ) had the highest control efficacy and were the most effective against T. flavus after 7 days of treatment. EOs are complex mixtures of secondary plant metabolites with a significant chemical variability [45,46]. Therefore, the individual or combined properties of the Rutaceae EOs tested against T. flavus could potentially be used to develop alternative pesticides for crop protection.

Discussion
In agricultural pest control, pesticide resistance is becoming increasingly prominent; thus, a higher demand exists for developing and applying efficient and safe insecticides [5]. Plant essential oils can be used in pest thrips management strategies and reduce the overuse of synthetic pesticides [44]. The present study was conducted to evaluate the insecticidal activity of five Rutaceae EOs for T. flavus control, followed by a preliminary identification of their chemical composition. Among the EOs tested, orange leaf oil (LC 50 = 0.26 g/L), zanthoxylum oil (LC 50 = 0.27 g/L), and pomelo peel oil (LC 50 = 0.44 g/L) showed the highest gastric toxicity. The pot experiments' results demonstrated that orange leaf oil (93.06 ± 3.67% at 540.00 g a.i.·hm −2 , 97.22 ± 1.39% at 720 g a.i.·hm −2 , 100.00% at 900.00 g a.i.· hm −2 ), zanthoxylum oil (98.73 ± 1.27% at 900 g a.i.· hm −2 ), and pomelo peel oil (100.00% at 900 g a.i.·hm −2 ) had the highest control efficacy and were the most effective against T. flavus after 7 days of treatment. EOs are complex mixtures of secondary plant metabolites with a significant chemical variability [45,46]. Therefore, the individual or combined properties of the Rutaceae EOs tested against T. flavus could potentially be used to develop alternative pesticides for crop protection.
As we emphasized the control efficacy of the EOs against T. flavus, only a preliminary chemical analysis of the EOs was performed ( Figure S1). The chemical composition of the tested EOs was, in part, consistent with previous reports [30,[47][48][49]. Limonene is the major constituent of EOs extracted from citrus by-products, whereas linalool, myrcene, sabinene, and pinene are characteristic of the Rutaceae EOs [48][49][50]. However, certain EOs' constituents differed from those reported in other studies. For example, the analysis of essential oil from the orange peel parts of C. sinensis from Argentina identified limonene (90.48%) as the most prominent compound [49]. The main constituents of Zanthoxylum bungeanum essential oil, extracted using a low-eutectic solvent and steam distillation, were terpineol-4-ol (13.13%) and (-)-β-pinene (11.17%) [51]. Many studies have shown that differences in the prominent essential oil compounds could be explained by variables such as harvest time, plant ontogeny, geographical location, plant parts, as well as extraction techniques [52][53][54][55][56].
Therefore, we suggest that the insecticidal activity of pomelo peel oil, orange leaf oil, and zanthoxylum oil may be attributed to their main constituents, such as D-limonene (78.36%), methyl jasmonate (51.50%), and linalool (52.69%), individually, or synergistically to the other active compounds that are present in lower concentrations [16]. Previous studies have also shown that these Rutaceae EO compounds are known for their insecticidal activity against many insects [57][58][59][60][61]. For example, the main compound of Protium heptaphyllum (Aubl.) (Sapindales: Burseraceae), D-limonene, exhibited contact and fumigation toxicity and had an ovicidal activity on C. maculatus (Coleoptera: Chrysomelidae: Bruchinae) [57]. D-limonene from celery (Apium graveolens L.) seeds and roots exhibited contact toxicity against T. castaneum, Lasioderma serricorne (Coleoptera: Anobiidae), and Liposcelis bostrychophila (Psocoptera: Liposcelididae) with LD 50 values of 14.57 µg/adult, 13.91 µg/adult, and 810.85 µg/cm 2 [58]. D-limonene exhibited contact toxicity (ranging from 132.48 to 828.79 µg/cm 2 ) and fumigant toxicity (LC 50 = 4.55 mg/L, 7.92 mg/L) against S. zeamais and Sitophilus oryzae L. [59]. Linalool and D-limonene exhibited excellent fumigant toxicity against red imported fire ants (Solenopsis invicta Buren). Linalool resulted in a 100% mortality rate against red imported fire ant workers at 5, 10, and 20 mg/tube after 8 h of treatment, and D-limonene induced >86% mortality after 8 h of exposure [60]. Linalool demonstrated both contact and fumigant activities against L. serricorne (LD 50 = 15.36 µg/larva) [61]. In addition, the insecticidal activity of the EOs tested depends on the plants they derive from, their concentration, and exposure time [16]. Two common compounds of citrus oil and pomelo peel oil were D-limonene and beta-myrcene, but the control efficacy of the two EOs against T. flavus differed considerably. This suggests that alpha-pinene (0.89%), sabenene (0.56%), or other compounds may antagonistically affect essential oil toxicity. One noteworthy observation was the lower activity of the citrus oil in samples with a higher relative percentage. The toxic effect of the EOs was due to the combined activities of different components, either with or without significant individual component toxicity against the target pests, e.g., the EO constituents of Ocimum kilimandscharicum and Ocimum kenyense (Labiateae) on S. zeamais and Rhyzopertha dominica and the EO of Piperaceae plants on Callosobruchus chinensis (L.) [62,63].
Both zanthoxylum oil and Chuan-shan pepper oil were also attractive to T. flavus in the olfactometer test. The GC-MS analysis results revealed linalool as the main component of the two EOs, which may be the key signaling compound that attracts T. flavus. Linalool is a common volatile organic compound that mediates host selection, foraging behavior, and other insect life activities. The attractiveness of linalool to thrips or other insects is supported by previous studies reporting the attraction of Thrips major Uzel to Sambucus nigra L. (Caprifoliaceae) [64], Thrips obscuratus (Crawford) to Lonicera japonica (Thunberg) (Caprifoliaceae) [65], and of F. occidentalis to synthetic linalool [66,67]. As a secondary component of the two EOs, D-limonene was repellent to Bemisia tabaci (Gennadius) and Tetranychus urticae Koch, whereas sabenene exposure resulted in no significant responses [68,69]. Beta-myrcene, on the other hand, was attractive to both males and females of Myllocerinus aurolineatus (Voss) (Coleoptera: Curculionidae) [70].
Developing a botanical pesticide is a lengthy process, starting with identifying plant sources with effective biocide activity and then identifying and characterizing its active ingredients [30,44]. This study focused on the insecticidal activity of five Rutaceae EOs against the target pest T. flavus and exploited their potential for a practical field application. The chemical composition of these EOs needs to be further evaluated by more advanced analytical chemistry techniques. Although the current insecticidal efficacy of the EOs is slower than that of faster-acting chemical insecticides, and the exact mechanism of action of the main chemical compounds is still not yet fully understood, they still have a good potential for development and applications [71,72]. As a single crop protection measure, spraying plant EOs may not be sufficiently effective [73,74]. However, their potential to control thysanopteran pests should be fully exploited in integrated pest-management strategies [16].
The injection port was operated in splitless mode with a constant helium flow of 1 mL·min −1 . The injector temperature was 230 • C, the ionization potential was 70 eV, and the scan frequency was 2 sec −1 . Following injection, the column temperature was maintained at 60 • C for 1 min, ramped at 11 • C·min −1 to 90 • C, held for 2 min, ramped at 2 • C·min −1 to 95 • C, held for 1 min, ramped at 18 • C·min −1 to 180 • C, held for 2 min, ramped at 2 • C·min −1 to 185 • C, held for 1 min, ramped at 13 • C·min −1 to 225 • C, held for 2 min, ramped at 2 • C·min −1 to 270 • C, and held for 1 min. The limit of detection (LOD) was approximately 2.0 ng ± 0.4 ng. The compound identification was conducted using the GC-MS solution software application, including the mass spectra library NIST 147 and NIST 27 (National Technical Information Services).

Laboratory Bioassay
The gastric toxicity was tested using the leaf-dipping method [76]. Serial dilutions of the five EOs (0.2, 0.4, 0.6, 0.8, 1.0 g/L) were prepared in acetone for evaluation. Fresh soybean leaves of uniform size, undamaged and free from pests and diseases, were selected, washed with water, and dried naturally. The leaves were immersed for 10 s in the tested EO solution and were subsequently removed. After natural drying, the leaves were placed in a centrifuge tube (50 mL, 30 mm diameter). Thirty adult thrips were added to the tube. The tubes were then quickly sealed with parafilm. Approximately 70 micropores were made in the parafilm, using as much force as possible with an insect pin (2#, 0.38 mm diameter), and the holes were evenly distributed. Thirty thrips were tested for each treatment concentration at three independent replicates. An acetone treatment was used as a control. All treated individuals were kept in incubators at 25 ± 1 • C and 70% ± 5%. Mortality was determined after 24 h. Thrips were considered dead if they did not react when touched with a writing brush [75].
According to the bioassay data, adjusted mortality was calculated as follows [76]: where MR is the mortality rate, ND is the number of dead insects, NA is the number of insects treated, AM is the adjusted mortality, MRT is the mortality rate of the treatment group, and MRC is the mortality rate of the control group.

Pot Experiments
Pot experiments were carried out in a greenhouse (25 ± 2 • C and natural lighting) to determine the effect of the five EOs on T. flavus control [7]. Soybeans were planted in batches in pots. A disease-and pest-free soybean seedling was selected and maintained in each pot during the second compound leaf development stage. The pot was then covered with a 200-mesh nylon netting. Based on the results of the laboratory bioactivity tests, serial dilutions of citrus oil and orange leaf oil (900.0, 1800.0, 2700.0, 3600.0, and 4500.0 g a.i.· hm −2 ), as well as pomelo peel oil, zanthoxylum oil, and Chuan-shan pepper oil (180.0, 360.0, 540.00, 720.0, and 900.0 g a.i.·hm −2 ) were prepared in acetone for evaluation, respectively. Each concentration was replicated 3 times, with the treatment without the EOs serving as the control. After spraying 5 mL evenly per pot with a spray bottle, the plants were allowed to dry naturally in a windless place. Thirty adult thrips were introduced per pot. The number of dead individuals was observed and recorded 1 day, 3 days, and 7 days after spraying.
According to experimental data, the control efficacy in pot experiments was calculated as follows: where CE is the control efficacy; AT is the number of insects in the treatment group after treatment with EOs; BC is the number of insects in the control group before treatment with EOs; BT is the number of insects in the treatment group before treatment with EOs; and AC is the number of insects in the control group after treatment with EOs.

Olfactory Test
The attractant/repellent activity of each essential oil to individual T. flavus adults was measured using a Y-tube olfactometer, as described by Zhang et al. [77]. The essential oil was tested with a 1 µL volume dropped on filter paper in the odor chamber. Pure humidified air was used as a control. Fluorescent light was set in parallel above the Y-tube to avoid light interference. The average intensity of illumination above the olfactometer was 7800-8000 lux. Both arms of the tube were filled with pure humidified air at a rate of 300 mL/min. A single T. flavus adult was placed at the entrance of the olfactometer, and after 5 min, its position in the tube was recorded [78]. The response criteria of T. flavus adults were determined as follows: if the adults climbed to more than half the length into one of the tubes and remained for 1 min or more, it was deemed that the adults chose this odor source; if the adults kept still or made no choice after 5 min, it was deemed as a no choice. At least thirty adults were exposed to each essential oil treatment. After the evaluation of five adults, the olfactometer tube was cleaned with acetone (99.5%, Xintong Fine Chemical Co., Ltd., Tianjin, China) and then heated at 100 • C for 30 min. All bioassays were conducted between 9:00 and 15:00 h in a room whose temperature was maintained at 24-27 • C.

Statistical Analysis
The data analysis was carried out using Data Processing System (DPS) software version 13.5, http://www.dpsw.cn/dps_eng/index.html (accessed on 5 March 2023) [78]. A oneway ANOVA analysis was used to evaluate the significance of the differences in adjusted mortality between different EOs applied. Tukey's HSD test was used at a 5% significance after an arcsine transformation of the adjusted mortality data. The analysis of the median lethal concentration (LC 50 ) resulting from the treatment with EOs was determined by a log-probit analysis with 95% fiducial limits. Differences between the number of T. flavus adults entering each arm of the olfactometer for each paired treatment were analyzed using the χ 2 test. All figures were prepared using GraphPad Prism 8.0 software (GraphPad Software, Boston, MA, USA).

Conclusions
The results demonstrated that under controlled conditions, orange leaf oil, zanthoxylum oil, and pomelo peel oil had strong insecticidal activities. Thus, they have the potential to be utilized as effective and low-toxicity botanical insecticides and synergistic agents against T. flavus. Further studies are needed to develop formulations based on the bioactive molecules of these EOs to be used as novel, more effective, and sustainable biopesticides for T. flavus control.