Determination of fumigant toxicity of single, binary and tertiary mixtures of three essential oils against Frankliniella occidentalis Pergande (Thysanoptera: Thripidae)

In this study, three essential oils extracted from sage (Salvia officinalis L.), thyme (Thymus vulgaris L.) and daphne (Laurus nobilis L.) were used individually against the adult Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) pest. Afterward, the fumigant toxicity of binary (1:1) and tertiary (1:1:1) mixtures was investigated under laboratory conditions. The results showed that all essential oils tested, both alone and in all binary and tertiary mixtures, had fumigant toxicity on adult F. occidentalis. Mortality rates of 3–5-day-old adults of F. occidentalis increased with increasing concentration from 2 to 10 µL/L. In the entire study, binary and tertiary mixtures of the oils were more successful than the individual application. At a dose of 10 µL/L, all binary and tertiary mixtures of the oils caused 100% mortality at all exposure times. However, when sage oil was fumigated for 24 h and applied alone, the mortality rate was less than 100%. Moreover, the highest synergistic effect was found in tertiary essential oil mixtures (sage + daphne + thyme) with 10.0 µL/L air LC50 value in all application doses, counted after 48 h. Our overall results show that binary and tertiary mixtures of all tested essential oils are promising candidates for protection against western flower thrips.


Introduction
Destructive insects, which are one of the most important factors limiting agricultural production, cause significant product losses.Among them, the important quarantine pest Frankliniella occidentalis (Thysanoptera: Thripidae) is a species that causes serious damage in the world and in Turkey.This polyphagous species has a wide range of hosts such as vegetables, cotton and ornamental plants (Bielza 2008;Wu et al. 2021).In Turkey, complaints about pests in greenhouses, especially in pepper greenhouses, are increasing day by day (Tunç and Göçmen 1995;Yıldırım and Başpınar 2013;Kamış and Dağlı 2022).
The high doses and frequent sprayings applied by the farmers have led to an increase in the resistance that has developed since the 1980s, and the resistance of many insecticides against F. occidentalis has been determined until today (Cloyd 2016;Dağlı 2018).Since the pest in question gives a large number of offspring during the year, resistance to insecticides can occur in a short time (Reitz et al. 2020).This situation leads to intensive chemical applications and harms the environment and the natural enemy population (Bielza 2008;Atanasova et al. 2022).Therefore, there is a need for integrated pest management (IPM) programs.
In recent years, plant extracts and essential oils have started to be seen as an alternative to synthetical pesticides in the management of agricultural pests.The use of herbal extracts as insecticides dates back to ancient times.Helleborus plant (Christmas rose flower) has been used as a natural insecticide in Roman times (Smith and Secoy 1975).These substances, which are being investigated with increasing interest, have been found to have insecticidal, repellent, anti-feedant and growth inhibitory, and egg-laying inhibitor effects against pests (Jannet et al. 2001;Sampson et al. 2005;Kumral et al. 2010;Taş 2011).In addition, it has been reported that the contact effect of herbal extracts against many pests is lethal (Karakoç and Gökçe 2012;Taş et al. 2015).
Similarly, the use of plant-derived essential oils as insecticides has been one of the important research topics.High lipophilic (water-insoluble) essential oil components can dissolve in fatty tissues when taken into the body of the pest due to these properties, making the control even easier (Tsao et al. 1995).It has been determined that herbal oils have different effects against pests such as toxicity, repellency, nutritional inhibition, growth and reproduction inhibition (Shaaya et al. 1993;Ndungu et al. 1995;Isman 2000).Many studies have been conducted on essential oils in recent years (Isman 2015;Pavela and Benelli 2016).Essential oils are complex combinations of simple aromatic terpenes extracted from aromatic plants using hydrodistillation (Isman 2015;Pavela and Benelli 2016).Many studies have shown that essential oils have pesticidal and insecticidal properties (Isman 2015;Benelli et al. 2018;Benelli and Pavela 2018a, b;Pavela et al. 2019).Given the ease with which EOs penetrate into the environment, they can be regarded acceptable active ingredients for botanical pesticides fumigated in enclosed locations such as storage facilities or greenhouses (Koschier 2008;Stepanycheva et al. 2018;Mejdoub et al. 2019).
In the last three decades, significant research has been conducted on the development of new botanical insecticides as an alternative to existing synthetical insecticides (Çetin et al. 2004;Erler 2004;Erler et al. 2007;Isman 2015;Benelli et al. 2018;Benelli and Pavela 2018a, b;Pavela et al. 2019;Stepanycheva et al. 2019;Gharbi and Tay 2022).Many essential oils and their components have been reported to have fumigant activity on thrips (Sedy and Koschier 2003;Stepanycheva et al. 2019;Gharbi and Tay 2022).This study was carried out to determine the fumigant toxicity of some aromatic plant essential oils and mixtures against F. occidentalis, which is the main pest of many ornamental plants and vegetables.

Plant materials and essential oil extraction
Dry parts of sage (Salvia officinalis L.), thyme (Thymus vulgaris L.) and daphne (Laurus nobilis L.) were used for extraction.The collected plant materials were first dried naturally on laboratory benches for 5-7 days at room temperature (23-24 °C) until crisp and then hydro-distilled to extract their essential oils.Taxonomic identification was made by subject experts in the Department of Food Technology and Medicinal and Aromatic Plants, Batı Akdeniz Agricultural Research Institute (BATEM), where the samples were stored.
The essential oil composition of samples was analyzed by gas chromatography (Agilent 7890A) coupled by flame ionization detector and mass spectrometry (Agilent 5975C) using capillary column (HP Innowax Capillary; 60.0 m × 0.25 mm × 0.25 µm).Essential oils were diluted in a 1:50 ratio with hexane.GC-MS/FID analysis was carried out at split mode of 40:1.Injection volüme and temperature were adjusted as 1 µL and 250 °C, respectively.Helium was the carrier gas at a constant flow rate of 0.8 mL/min.The oven temperature was programmed as follows: 60 °C for 10 min, increased at 4 °C/min to 220 °C, and held at 220 °C for 10 min.MS spectra were monitored between 35 to 450 amu and the ionization mode used was electronic impact at 70 eV.The relative percentage of the components was calculated from GC-FID peak areas, and components were identified by Wiley 7 and Oil Adams Libraries (Uysal Bayar and Çınar 2020).The chemical compositions of the essential oils used within the scope of this research are given in Table 1.

Insect material
Bean fruits were used to provide a feeding and egg-laying for thrips.Plastic containers with the lids, of which were covered with filter paper, were used as production cages and 2-3 layers of towel paper were laid on the bottom to allow the thrips to pass into the pupa stage.Bean fruits, which were disinfected with bleach and dipped in sugar and amino acid solutions and dried, were left in the production cages with the thrips desired to be produced.Furthermore, the green bean fruits were replaced with new ones at 3-4 day intervals.The thrips were reared in a climate chamber with a temperature of 24 ± 1ºC and a day length of 16:8 h (light: dark).Adult insects aged 3-5 days were used for fumigant toxicity tests.All trials were carried out under the same conditions.

Fumigant toxicity tests
The effectiveness of the tested essential oils as a fumigant against adults of F. occidentalis was carried out in the laboratory of the Plant Health Department of Batı Akdeniz Agricultural Research Institute (BATEM).Fumigant toxicity tests were carried out according to the method described by Erler (Erler 2005(Erler , 2007)).
To determine the fumigant toxicity of essential oils and their binary and tertiary mixtures, desiccators with a net volume of 10 L were used.Four replicates were used for each dose and combination.Each dose was applied with an automatic pipette on a blotter (9 cm in diameter) adhered to the underside of the desiccator lid.Then, the Vaseline-applied desiccator lids were tightly closed in an air-tight manner.
Preliminary tests were performed to determine the appropriate dose and dose ranges.The application doses were determined as 2, 4, 8 and 10 µL/L air.The binary dose mixtures of the tested essential oils were prepared in the ratio of 1:1 and the tertiary mixtures were prepared in the ratio of 1:1:1.The experiment was initiated by placing the desiccators under controlled conditions (26 ± 1 °C temperature, 60 ± 5% proportional humidity).No material was applied to the control desiccator.In agricultural practices, the maximum closed period of greenhouses was approximately 8 h.Therefore, in this study, the fumigation time was chosen as 8 h, taking into account the agriculture practices.At the end of this period (8 h), the desiccators were opened to remove the fumigation effect and the petri dishes were kept in an open environment for 48 h.During this period, dead and live individuals were counted at 8, 24 and 48 h to reveal the continuing effect of the treatment.Insects were considered dead when no leg or antennae movement was observed when poked with a fine brush.

Determination of Bu sebzeler en çok ekilen ve trips zararından etkilenenlerdir.of essential oils
To determine the phytotoxic effects of essential oils on vegetables, the most commonly grown tomato, eggplant, cucumber and bean cultivars in Antalya region were selected.Thus, Erciyes cultivar for pepper, Phaselis F 1 for eggplant, PTK 40 for cucumber and Özayşe for beans were used.The plants were grown in climate chamber with 26 ± 1C° temperature, 65 ± 5% RH and an 18:6 lighting period.Each seedling was transplanted into 12 × 10 cm pots containing a sterile mixture of peat:perlite (2:1).When the plants reached approximately 15 cm (3-4 leaves) in length, they were placed in desiccators as in other fumigant effect trials, and the highest doses for each essential oil (S.officinalis: 10 µl/l; T. vulgaris: 10 µl/l; L. nobilis: 10 µl/l; each dose was prepared as 1:1 in binary and 1:1:1 in the tertiary mixtures of the tested essential oils) was sucked onto the blotting paper (9 cm in diameter) adhered to the inside of the desiccator lid with the help of a micropipette, and the desiccator lid was closed without delay and fumigation was done for 8 h.At the end of the fumigation, the plants removed from the desiccators were observed for 48 h, and the signs of phytotoxicity were recorded.The experiments were carried out with 5 replications for each plant as control and essential oil.Phytotoxic effects of essential oils tests were carried out according to the method described by Topuz et al. (2018).

Data analysis
The mortality rates obtained from the trials were analyzed using the SPSS program after calculating adjusted mortality rates with the Abbott formula, and statistical differences between the means were tried to be revealed with the DMRT (P ≤ 0.05).While determining the fumigant effect, the data obtained from the adult effects trials were calculated as LC 50 , LC 90 values at a 95% confidence interval according to the probit analysis method (POLO-PC, LeOra Software, 1997, Berkeley, CA, USA).

Results
One of the important effects of essential oils on pests is the fumigant effect.The chemical compositions of the essential oils used in this research are given in Table 1.
The analysis of volatile oil components revealed interesting findings in daphne, thyme, and sage essential oils.Daphne essential oil contained a total of 14 components, with the main constituents being 1,8-cineole, alpha-terpinyl acetate, sabinene, and alpha-pinene, accounting for 57.49, 9.65, 9.23, and 6.46%, respectively.Thyme oil, on the other hand, exhibited eight components, and the primary compounds identified were carvacrol (93.95%), linalool (1.60%), and thymol (1.48%).Lastly, sage oil displayed the highest complexity with 20 components, and its main constituents were alpha-pinene (25.85%), 1,8-cineole (17.81%), camphor (13.87%), and camphene (10.07%).More than 80% of the components in laurel and sage essential oils consist of monoterpenes.The dominant compound in daphne essential oil is 1,8-cineole, followed by alpha-terpinyl acetate and sabinene.When analyzing the components of thyme essential oil, the highest volatile compound identified was carvacrol, accounting for 93.95%.In sage essential oil, the predominant component was alpha-pinene, followed by 1,8-cineole, camphor, and camphene.Also in this study, the fumigant effects of different concentrations of essential oils of F. occidentalis on adults were investigated (Table 2).Fumigation was applied to the adult stage of the pest for 8 h.Adults were counted as dead and alive in different periods until the 48th h; LC 50 and LC 90 values of essential oils were calculated from the data obtained (Tables 2, 3) (Table 4).
All essential oils tested, both alone and in binary and tertiary mixtures, showed differential fumigant toxicity to F. occidentalis adults at all dose × exposure time combinations tested (Tables 2 and 3).Overall, mortality increased with increasing dose from 2 to 10 µL/L air and with increasing count time from 24 h to 48 h.
Among the essential oils tested, thyme and daphne oils were the most successful, achieving 100% mortality at the highest dose (10 µL/L air) at the 24th and 48th-h counts (Table 2).In contrast, daphne oil at the highest dose (10 µL/L air) had lower mortality rates than daphne and thyme.Moreover, thyme and daphne showed a similar high effect (Table 2).
Compared to the single-applied essential oils, the mortality rates of the binary and tertiary mixtures of essential oils were higher.In the 24th and 48th-h counts of thyme + daphne oil mixtures, 100% mortality was achieved at the highest dose (10 µL/L air) (Table 3).In the 48th h count, a high mortality rate (99.9%) was determined in the thyme + sage mixture, while a 72.8% mortality rate was determined (10 µL/L air) in the daphne + sage at the highest dose.In addition, the tertiary mixture of these Table 2 Mean percent mortality of 3-5-day-old adults of Frankliniella occidentalis exposed to the vapors of three essential oils for various dose x exposure time combinations *In each treatment, means within a row followed by the same capital letter are not significantly different (DMRT, P ≤ 0.05) **In each treatment, means within a column followed by the same lower case letter are not significantly different (DMRT, P ≤ 0.05)  4 is examined, the LC 50 value calculated from the data obtained from the 8th-h counts was found to be 2.9 µL/L for thyme essential oil, 13.9 Table 3 Mean percent mortality of 3-5-day-old adults of Frankliniella occidentalis exposed to the vapors of binary and tertiary mixtures of three essential oils for various dose x exposure time combinations *In each treatment, means within a row followed by the same capital letter are not significantly different (DMRT, P ≤ 0.05) **In each treatment, means within a column followed by the same lower case letter are not significantly different (DMRT, P ≤ 0.05) 1.5 (0.9-2.0) 6.8 (5.5-9.5)1.9 ± 0.1 24 1.3 (0.7-1.7) 5.2 (4.3-6.9)2.1 ± 0.1 48 1.0 (0.5-1.4) 4.4 (3.6-5.6)2.0 ± 0.1 µL/L for sage and 3.4 µL/L for daphne.The LC 50 values calculated from the data obtained from the 8th-h counts in the oil mixtures were 2.7 µL/L for the thyme + sage, 10.3 µL/L for the daphne + sage, 2.1 µL/L for the thyme + daphne and 1.5 µL/L for the thyme + sage + daphne.These values decreased gradually in the following counting periods and decreased to 2.0 µL/L for thyme essential oil, 12.2 µL/L for sage and 2.4 µL/L for daphne at the end of the 48th h.For the mixed oils, these values decreased gradually in the following counting periods and were determined as 1.9 µL/L for thyme + sage, 5.1 µL/L for daphne + sage, 1.3 µL/L for thyme + daphne and 1.0 µL/L for thyme + sage + daphne.
The LC 90 values were similar to LC 50 values, and a decrease in LC values was observed in all oils and oil mixtures in general with increasing counting hours.This situation was reflected in the probit analysis results.

Determination of phytotoxic effects of essential oils
Observations were made for phytotoxicity in plants until the 48th h after the 8-h fumigation process, and no phytotoxic effects were found on tomato, eggplant cucumber and bean plants with single, binary and tertiary mixtures of three essential oils.In general, no adverse events were observed in all control group plants kept under the same conditions as treated plants but not subjected to essential oil fumigation.

Discussion
The use of plant essential oils is an alternative to synthetical insecticides for thrips control and poses a low risk to human health (Sedy and Koschier 2003;Stepanycheva et al. 2019;Gharbi and Tay 2022).Binary mixtures of essential oils from different plant species can increase their toxic effects against pests (Benelli et al. 2017).In the present study, the mixture of essential oils of daphne, thyme and sage was determined to be toxic to the adult female stage of F. occidentalis.
The effectiveness of many essential oils as a fumigant to control F. occidentalis has been investigated and it has been observed that this insect is sensitive to some plant-derived chemicals.However, although there are existing publications on the fumigant activity of the essential oils tested in the current study against F. occidentalis, the effect of these oils individually and in their mixtures against the pest was determined for the first time.
In their study, Koschier et al. (2000) aimed to investigate the responses of adult female Western flower thrips to plant volatiles at various concentrations using Y-tube olfactometry.The researchers found that benzenoids benzaldehyde, p-anisaldehyde, and o-anisaldehyde were particularly attractive to the Western flower thrips.Additionally, other compounds such as p-anisaldehyde, nerol, ethyl nicotinate, and (E)-b-farnesene elicited positive responses at different concentrations.These findings suggest that specific plant volatiles can be used to attract Western flower thrips, which could be useful in developing strategies for controlling their populations in agricultural settings.Understanding the chemical cues that attract Western flower thrips may also lead to the development of more targeted and environmentally friendly pest management approaches.Sedy and Koschier (2003) conducted a study to test the effects of two components of essential oils, carvacrol, and thymol, on the feeding activity, oviposition, and oviposition rate of F. occidentalis.Unlike our study, which examined the fumigation effect of the oils, Sedy and Koschier investigated the contact effect by spraying a commercial preparation containing carvacrol and thymol with a spray-tower.The results of their study showed that both components had an anti-ovipositant effect on F. occidentalis.In our study, we found that thyme oil had a high lethal effect on F. occidentalis, both alone and in mixtures with its high content of carvacrol (93.95).These findings suggest that thyme oil, particularly in combination with high levels of carvacrol, may be a promising tool in controlling the populations of Western flower thrips.The potential of essential oils as natural and safe alternatives to conventional insecticides warrants further investigation for sustainable pest management practices.
Several studies have investigated the effects of different essential oils and their components on the F. occidentalis.Stepanycheva et al. (2019) examined the acute toxicity results of 15 commercial essential oils applied by fumigation and the effect of sublethal concentrations on the fertility of F. occidentalis females.They found that Thymus mastichina species thyme had an LC 50 value of 3.6 mg/L and an LC 90 value of 4.6 mg/L.In contrast, our study determined the LC 50 value as 2.0 µL/L and the LC 90 value as 8.9 µL/L after 48 h of thyme application by fumigation.Allsopp et al. (2019) investigated the effectiveness of different carvacrol concentrations on F. occidentalis individuals through experiments on plum blossoms.They observed that 5, 1, and 0.1% concentrations of carvacrol significantly reduced the oviposition rate.In our study, four different concentrations of thyme oil were used, and the results showed mortality rates of 56.8, 63.3, 86.5, and 100% at doses of 2, 4, 8, and 10 µL/L, respectively, after 48 h of application.These findings suggest that essential oils, such as thyme oil and carvacrol, could be potential alternatives to conventional insecticides in controlling F. occidentalis populations.However, further research is necessary to assess the long-term effects of essential oils on the environment and their impact on nontarget organisms.
Several studies have investigated the effects of different essential oils and their components on F. occidentalis.Picard et al. (2019) found that thyme oil was one of the most effective repellents against F. occidentalis, which is consistent with the results of our study.Stepanycheva et al. (2018) showed that essential oils of Acorus calamus, Juniperus virginiana and Melissa officinalis had a repellent effect on F. occidentalis larvae.Gharbi and Tay (2022) found that (R)-linalool was more toxic than (S)-linalool against F. occidentalis adults, but since the content of linalool was low in the plants we tested, it is unlikely to have contributed significantly to the mortality rate in our study.Janmaat et al. (2002) found that the essential oil component p-cymene had a high mortality rate on F. occidentalis adults and larvae, but its contribution to mortality in our study was also likely to be low due to its low content in the plants we tested.Overall, these studies provide valuable information on the potential use of essential oils and their components as natural pest control agents for F. occidentalis.

Conclusion
After considering the findings of the study, it can be concluded that essential oils have shown potential as effective natural insecticides for controlling F. occidentalis when applied via fumigation.According to the findings, binary and tertiary essential oil combinations were more active than individual oils (Tables 2 and 3).This indicates a possible synergism, and the usage of combinations of plant essential oils may be more effective than individual oils in preserving plants.Observing the effect of essential oils (EOs) on thrips is essential to keep the pest population below the economic harmfulness threshold.Results in terms of fumigation were promising and no immediately phytotoxicity was observed.However, further tests will have to be done to explain the mechanism of action of this phenomenon, and the impact of such EOs on non-target organisms will have to be tested as well.

Table 1
Chemical composition of investigated essential oil samples *HP Innowax Capillary; Linear retention index on a 60.0 m × 0.25 mm × 0.25 µm column was determined experimentally using homologous C8-C40 alkane series The LC 50 and LC 90 values calculated from the counts of live/dead individuals at different times after fumigation of F. occidentalis adults with different concentrations of essential oils are given in Table4.When Table oils (thyme + sage + daphne) was more successful than all binary mixtures (Table3).