doi:10.14720/aas.2020.115.2.1317 Original research article / izvirni znanstveni članek Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly alternative to the synthetic insecticides in management of two stored-products insect pests Asgar EBADOLLAHI 1 2 Received October 29, 2019; accepted March 22, 2020. Delo je prispelo 29. oktobra 2019, sprejeto 22. marca 2020 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly alternative to the synthetic insecticides in management of two stored-products insect pests Abstract: The lesser grain borer [Rhyzopertha dominica (Fabricius, 1792)] and the red flour beetle [Tribolium castaneum (Herbst, 1797)] are among the cosmopolitan damaging pests on several stored-products. The overuse of chemical pesticides in the control of such pests caused several side-effects including environmental contaminations, human health problems, and insect pests' resistance. In this circumstance, researchers have focused on safe and effective alternatives to chemical pesticides. In the present study, the insecticidal efficiency of essential oil extracted from the summer savory (Satureja hortensis L.) was assessed on the R. dominica and T. castaneum adults. The chemical profile of essential oil was evaluated through a gas chromatography-mass spectrometer, in which estragole, p-ocimene and d-limonene were the main components. The essential oil had considerable fumigant toxicity on insect pests. The mortality of insects was dependent on the essential oil concentration and exposure time. Probit analysis indicated that R. dominica with low LC50 values (Lethal Concentration to kill 50 % of tested insects) was more susceptible than T. castaneum. Accordingly, S. hortensis essential oil with a high level of phe-nylpropanoid and terpenic compounds can be recommended as an efficient and natural alternative to the detrimental chemicals in the management of R. dominica and T. castaneum. Key words: essential oil; estragole; Satureja hortensis; fumigation; coleopteran pests Na estragolu bogato eterično olje vrtnega šetraja (Satureja hortensis L.) kot okolju prijazna alternativa sintetičnim insek-ticidom pri zatiranju dveh vrst skladiščnih škodljivih žuželk Izvleček: Žitni kutar [Rhyzopertha dominica (Fabricius, 1792)] in rižev mokar [Tribolium castaneum (Herbst, 1797) sta kozmopolitski vrsti škodljivcev, ki povzročata škodo na mnogih uskladiščenih pridelkih. Prekomerna raba insekticidov pri zatiranju takšnih škodljivcev ima številne stranske učinke, vključno z onesnaževanjem okolja, zdravstvenimi problemi ljudi in odpornostjo škodljivih žuželk. V tej raziskavi so se raziskovalci osredotočili na varno in učinkovito alternativo sintetičnim in-sekticidom. Insekticidna učinkovitost eteričnega olja iz vrtnega šetraja (Satureja hortensis L.) je bila preizkušena na odraslih osebkih obeh vrst zgoraj omenjenih škodljivcev. Kemična sestava eteričnega olja je bila ovrednotena s plinskim kromato-grafom in masnim spektrometrom, ugotovljeno pa je bilo, da so estragol, p-ocimen in d-limonen glavne sestavine. Zaplinje-vanje z eteričnim oljem je imelo znaten toksični učinek na škodljivi žuželki. Smrtnost žuželk je bila odvisna od koncentracije eteričnega olja in časa izpostavitve. Analiza Probit je pokazala, da je vrsta R. dominica z manjšimi LC50 vrednostmi bolj občutljiva kot vrsta T. castaneum. Glede na to bi lahko eterično olje iz vrtnega šetraja z veliko vsebnostjo fenilpropanoidov in terpenov priporočili kot učinkovito in naravno alternativo škodljivim kemikalijam pri zatiranju omenjenih škodljivcev. Ključne besede: eterično olje; estragol; Satureja hortensis; zaplinjevanje; škodljivi hrošči 1 University of Mohaghegh Ardabili, Moghan College of Agriculture and Natural Resources, Ardabil, Iran 2 Corresponding author, e-mail: ebadollahi@uma.ac.ir Acta agriculturae Slovenica, 115/2, 307-314, Ljubljana 2020 115-2 vsebina.indd 307 23. 06. 2020 07:25:24 A. EBADOLLAHI 1 INTRODUCTION 2 MATERIALS AND METHODS Secondary metabolites announce the evolution of chemical defenses in plants which are often formed as by-products throughout the production of primary metabolites. Secondary metabolites have several essential roles especially in the protection against herbivores and in the attraction of pollinators' (Dinan, 1995; Bohinc et al., 2012). Plant-derived essential oils as well-known secondary metabolites can be produced in several aerial parts including leaves, flowers, seeds, stems and the roots of aromatic plants. Essential oils are generally composed of isoprene units as terpenes and phenylpropane (Bak-kali et al., 2008). Although terpenes such as monoter-penes (2 units of isoprene, C10), sesquiterpenes (3 units of isoprene, C15), and diterpenes (4 units of isoprene, C20) have a high quantity, the monoterpenoids (oxygenated monoterpenes) are often the most components of the many essential oils (Breitmaier, 2006; Abdel-Tawab, 2016). Along with the application of essential oils in the perfumery and pharmaceutical industries, their lethal and sub-lethal effects especially fumigant toxicity of essential oils have been approved toward different class and orders of main insect and acari herbivores (Regnault-Roger et al., 2012; Rojht et al., 2012; Ebadollahi & Jalali-Sendi, 2015). Summer savory [Satureja hortensis L. (Lamiaceae)], as an aromatic spice and food preservative, widely distributed and/or cultivated in many countries. It used in Iranian traditional medicine to treat intestinal and stomach disorders such as indigestion and diarrhea, muscle pain, thrombosis, and cardiovascular diseases (Hajhashemi et al., 2000; Yazdanparast et al., 2008). Moreover, along with antibacterial, antifungal, antioxidant, and cytotoxic activities of S. hortensis, its potential on the insect pest management have also been documented (Mahboubi & Kazempour, 2011; Miladi et al., 2013; Gombac & Trdan, 2014; Farzaneh et al., 2015; Ghorbanpour et al., 2016). R. dominica (lesser grain borer) and T. castaneum (red flour beetle) are among the cosmopolitan serious pests of stored-products such as cereal and legume grains, dried fruits, spices, flours, leather, and even packaging materials made from wood and paper. Further, the quality of infested products strongly reduces due to the residues of insect bodies and their unpleasant smell (Villaverde et al., 2007; Edde, 2012). As part of a program aimed at studying the insecti-cidal activity and chemical composition of plant essential oils, we have assessed the fumigant toxicity and chemical profile of S. hortensis essential oil against R. dominica and T. castaneum. Hope the range of introduced active bioagents derived from aromatic plants has extended by the results of the present study. 2.1 ESSENTIAL OIL EXTRACTION AND ANALYSIS Fresh 10 cm aerial parts from the shoots of S. hortensis were sampled for essential oil extraction. The specimens were collected during April and May 2019 from Parsabad region (Latitude: 39°38' N, Longitude: 47°52' E, and height: 52 m), Ardebil province, Iran. The samples were dried at room temperature within a week and then ground using an electric grinder. Fifty grams ground plant material was poured into a Clevenger apparatus equipped with a 1000 ml balloon. The essential oil was extracted within 3 h and the obtained oil was stored in a refrigerator at 4 °C. Chemical profile of the S. hortensis essential oil was assessed using a gas chromatographic system (Agilent model 7890B) equipped with the mass spectrometer detector (Agilent model 5977A) according to Ebadollahi et al. (2017): chromatographic separation was performed on the HP-5MS (5 % phenyl-methyl-polysiloxane) capillary column (30 m length, 0.25 mm internal diameter, and 0.25 ^m film thickness) with 70 eV ionization energy. The injected volume was 1.0 ^l with 280 °C temperature. The temperature program of the column was set from 50 to 350 °C. Helium (99.999 %) was used as a carrier gas at 1 ml minute-1. The component was identified by comparison of their mass spectra with those from Wiley's MS library (7th edition) and NIST (National Institute of Standards Technology) in the library. 2.2 TESTED INSECTS The adult insects of R. dominica were obtained from the colonies at the Department of plant protection, University of Mohaghegh Ardebili, Ardabil, Iran. The adult insects of T. castaneum were collected from contaminated wheat grains in the warehouses of Parsabad city (Latitude: 39°38' N, Longitude: 47°52' E, and height: 52 m), Ardabil province, Iran. Adult insects were separately released on wheat grains in the breeding container. Adult insects were removed 48 h later and grains with insects' eggs were kept in an incubator at 25 ± 2°C and 65 ± 5 % relative humidity in dark (Arnaud et al., 2005). Synchronized adult insects with 1 - 7 old-days were selected. 2.3 BIOASSAY The fumigation bioassay was done according to the study of Ebadollahi (2018): twenty adults of both insects 308 Acta agriculturae Slovenica, 115/2 - 2020 115-2 vsebina.indd 308 23. 06. 2020 07:25:24 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly ... of two stored-products insect pests were separately located in 340 ml fumigant chambers. The tested concentrations of essential oil, based on the preliminary experiments, were from 11.76 to 47.06 pl l-1 and from 21.00 to 55.15 pl l-1 for R. dominica and T. cas-taneum, respectively. The essential oil concentrations were poured on the 2 x 3 cm piece of filter papers which were sealed to the inside of the container lids and the lids were closed using parafilm. Experiments were conducted for control groups without adding essential oil concentration. Each treatment was repeated 4 times and the insects' mortality was documented after 24, 48 and 72 h intervals. 2.4 STATISTICAL ANALYSIS Variance analysis was used to assess the significant effects of essential oils' concentrations and the exposure times. To compare the effects of independent factors concentration and exposure time on the insects' mortality, the w2 comparison was used. Calculation of lethal concentrations (LC), lethal times (LT) and linear regression analysis along with heterogeneity of the data by a Chi-squared test were done using SPSS software version 24 (IBM, Chicago, USA). 3 RESULTS 3.1 CHEMICAL COMPOSITION OF ESSENTIAL oIL Chemical analysis of S. hortensis essential oil identified 17 components at 99.21 %, in which 83.02 % are phenylpropanoid constituents. Five different groups of terpenes were also recognized in the essential oil, in which the monoterpene hydrocarbons (15.38 %) had the highest amount followed by sesquiterpenoids (0.43 %), monoterpenoids (0.26 %), a sesquiterpene hydrocarbon (0.08 %), and a diterpene (0.04 %). Estragole (82.10 %) as Table 1: Chemical composition of the essential oil isolated from Iranian Satureja hortensis Compound Retention Time (minute) Formula and Classification Percentage a-Pinene 5.30 C10H MH 0.91 Camphene 5.57 C10H MH 0.04 Sabinene 6.03 C10H MH 0.06 ß-Pinene 6.09 C10H MH 0.09 ß-Myrcene 6.33 C10H MH 0.12 d-Limonene 7.08 C10H MH 2.25 ß-Ocimene 7.46 C10H MH 11.86 a-Terpinene 8.27 C10H MH 0.05 Rosefuran 8.43 C10H 4o M 0.08 Estragole 11.51 C10H 2o Ph 82.10 E,E-2,6-Dimethyl-3,5,7-octatriene-2-ol 11.54 C10H 6o M 0.07 Bornyl acetate 14.23 C12H 60o2 M 0.11 Methyl Eugenol 18.71 C11H 4o2 Ph 0.92 Germacrene-D 21.05 C15H SH 0.08 spathulenol 23.76 C15H 40 S 0.31 Caryophyllene oxide 23.89 C15H 40 S 0.12 Eicosane 32.78 C20H DH 2 0.04 MH: Monoterpene Hydrocarbon 15.38 M: Monoterpenoid 0.26 SH: Sesquiterpene Hydrocarbon 0.08 S: Sesquiterpenoid 0.43 DH: Diterpene Hydrocarbon 0.04 Ph: Phenylpropanoid 83.02 Total 99.21 Acta agriculturae Slovenica, 115/2 - 2020 309 115-2 vsebina.indd 309 23. 06. 2020 07:25:24 A. EBADOLLAHI Table 2: Results of the variance analysis of S. hortensis essential oil fumigation on the adults of R. dominica and T. castaneum after 24, 48 and 72-h exposure times Insect Source of Variation df F p-value w2 R. dominica Concentration 4 467.987 * <0001 22.516 Time 2 155.009 * <0001 3.713 Time x Concentration 8 1.594 0.154 0.057 T. castaneum Concentration 4 324.572 * <0001 17.793 Time 2 142.271 * <0001 3.884 Time x Concentration 8 1.106 0.377 0.012 Significant at a = 1% Figure 1: Concentration - mortality lines for fumigant toxicity of S. hortensis essential oil against the adults of R. dominica and T. castaneum after 24, 48 and 72-h exposure times a phenylpropanoid constituent had the highest amount and monoterpene hydrocarbons ^-ocimene (11.86 %), and dl-limonene (2.25 %) were in the next points (Table 1). 3.2 FUMIGANT TOXICITY Results of the fumigant toxicity indicated that essential oil of Iranian S. hortensis had considerable toxicity on the R. dominica and T. castaneum adults. The results of variance analysis were summarized in Table 2. Concentrations of essential oil and exposure times had statistically significant effects on the insects' mortality but 310 Acta agriculturae Slovenica, 115/2 - 2020 their interaction wasn't significant. Furthermore, based on the w2 values, among these factors, the effect of essential oil concentration was more effective. The calculated R2 values for concentrations-mortality correlation were 0.959, 0.935 and 0.940 for R. dominica and 0.956, 0.953 and 0.960 for T. castaneum after 24, 48 and 72-h exposure times, respectively. So, there is a direct correlation between the concentrations of essential oil and mortality of both insects (Figure 1). Probit analysis indicated the calculated LC50 values (lethal concentration to kill 50 % of tested insects) of essential oil were significantly decreased from 24 h to 72 h for both insects (Table 3). For example, the 24 h-LC50 value of essential oil with 95 % confidence limits was 27.212 115-2 vsebina.indd 310 23. 06. 2020 07:25:24 Estragole-rich essential oil of summer savory (Satureja hortensis L.) as an eco-friendly ... of two stored-products insect pests Table 3: Results of Probit analysis for fumigant toxicity of S. hortensis against the adults of R. dominica and T. castaneum Time LC50 (95 % confidence limits) x2 Insect (h) (Ml l-1) (df = 3) Slope ± SE Significance * R. dominica 24 27.212 (24.657 - 30.361) 3.893 2.740 ± 0.294 0.273 48 22.193 (20.140 - 24.385) 7.062 2.897 ± 0.298 0.070 72 16.466 (12.128 - 20.013) 5.830 3.321 ± 0.329 0.120 T. castaneum 24 38.908 (35.951 - 42.688) 3.425 3.386 ± 0.412 0.331 48 30.757 (28.377 - 33.070) 3.810 3.691 ± 0.419 0.283 72 25.747 (23.020 - 28.021) 2.745 3.506 ± 0.429 0.433 Concentration LT50 (95 % confidence limits) X2 Insect (Ml l-1) (h) (df = 1) Slope ± SE Significance * R. dominica 47.06 10.301 (2.944 - 16.210) 1.765 2.060 ± 0.515 0.184 T. castaneum 55.15 12.682 (5.479 - 18.103) 2.023 2.282 ± 0.503 0.155 Since the significance level is greater than 0.05, no heterogeneity factor is used in the calculation of confidence limits. The number of insects for (24.657 - 30.361) M l-1 which was decreased to 16.466 (12.128 - 20.013) M l-1 after 72 h. Further, according to Table 3, adults of R. dominica with low LC50 values were significantly susceptible than T. castaneum adults to the S. hortensis essential oil at all exposure times. The lethal times to kill 50 % of tested insects (LT50 values) are also shown in Table 3. At a high tested concentration of S. hortensis essential oil (47.06 ^l l-1), the LT50 value was 10.301 (2.944 - 16.210) h against R. dominica adults. This value for T. castaneum adults with a concentration of 55.15 M l-1 was calculated as 12.682 (5.479 - 18.103) h. 4 DISCUSSION The composition of S. hortensis essential oil have been investigated in the previous studies; carvac-rol (11.0 %), p-cymene (19.6 %), sabinene (4.4 %), y-terpinene (16.0 %), and thymol (28.2 %) were found as major compounds by Mahboubi and Kazempour (2011). Thymol, p-cymene, y-terpinene, and carvacrol were not detected in the present study but a trace of sabinene (0.06 %) was determined. In contrast, estragole and ^-ocimene as major components of present work were not detected in the study of Mahboubi and Kazempour (2011). Farzaneh et al. (2015) showed carvacrol (48.0 %), p-cymene (11.7 %), myrcene (2.5 %), a-pinene (2.5 %), y-terpinene (24.2 %) were the main components. From these constituents, myrcene (0.12 %) and a-pinene (0.91 %) with different amounts were recognized in the essential oil of present study. In the other study, Miladi et al. (2013) also revealed that monoterpenoids (59.11 %) were the main chemical class of S. hortensis essential oil which is parallel with our results but they announced other components such as carvacrol, ^-caryophyllene, p-cymene, and y-terpinene. In contrary, Mohammadhos-seini and Beiranvand (2013) showed that the monoter-pene hydrocarbons such as myrcene, a-pinene, ^-pinene, a-terpinene, and a-thujene had the highest amount in the S. hortensis essential oil. These differences in the chemical profile of S. hortensis essential oil in the present and above-mentioned studies can be due to the variations in some of the influential factors, such as geographical and growing conditions, drying and extraction methods, on-togenetic stages, and season (Sefidkon et al., 2006; Pfefferkorn et al., 2008; Rezvanpanah et al., 2011; Ghorban-pour et al., 2016). Insecticidal properties of S. hortensis essential oil were acknowledged in some recent studies; appropriate fumigant toxicity of this oil was proved against Mediterranean flour moth [Ephestia kuehniella (Zeller, 1879)], Indianmeal moth [Plodia interpunctella (Hubner, 1813)], and T. castaneum (Mollaei et al., 2011). The calculated 48 h-LC50 value for T. castaneum in this work (192.350 ^l l-1) is much higher than the corresponding LC50 in the present study (30.757 ^l l-1). In the study of Tozlu et al. (2011), the S. hortensis essential oil with a high amount of carvacrol, ^-caryophyllene, p-cymene, S-terpinene, and a-terpinene was very toxic against the broad bean weevil [Bruchus dentipes (Baudi, 1886)]. They concluded that the S. hortensis essential oil toxicity is directly related to its components. Along with the fumigant toxicity of S. hortensis essential oil, the contact toxicity, repellency, and disruption in the enzymes' activity were also described (Mollaei et al., 2011; Heydarzade & Moravvej, 2012; Magierowicz et al., 2019). The results of these studies indicated that S. hortensis essential oil has considerable Acta agriculturae Slovenica, 115/2 - 2020 311 calculation of LC values is 400 for each time. The number of insects for calculation of LT values is 240 for each concentration. 115-2 vsebina.indd 311 23. 06. 2020 07:25:24 A. EBADOLLAHI insecticidal activities against stored-product insect pests which are in accordance with our findings. Estragole or methyl chavicol, as two major compounds identified in the present study, is a GRAS (Generally Recognized As Safe) nominated material and approved for food procedure (De Vincenzi et al., 2000). Its name originates from "estragon" which is a French word of tarragon (Artemisia dracunculus L.) (Misztal et al., 2010). Along with cytotoxic and antimicrobial properties of estragole (Bagamboula et al., 2004; Andrade et al., 2015), toxicity of this compound has also been approved against some of damaging stored-product insect pests including T. castaneum, the rice weevil [Sitophilus oryza (Linnaeus, 1763)], the maize weevil [Sitophilus zeamais (Motschulsky, 1855)], the booklice [Liposcelis bostry-chophila, Badonnel, 1931], the cigarette beetle [Lasio-derma serricorne (Fabricius, 1792)], and the adzuki bean beetle [Callosobruchus chinensis (Linnaeus, 1758)] (Kim & Ahn, 2011; Wang et al., 2011; Kim & Lee, 2014; Guo et al., 2015). Furthermore, the insecticidal properties of other main components identified in the present study including d-limonene and ^-ocimene were also documented (Tripathi et al., 2003; Guo et al., 2015; Kang et al., 2018). Accordingly, the fumigant toxicity of S. hortensis essential oil may be attributed to such constituents. However, the existence of synergistic effects between other compounds is also possible. 5 CONCLUSION Synthetic pesticide residues can be found in different parts of our surrounding environment from water and soil to everybody's foods and even human breast milk samples (Damgaard et al., 2006; Nicolopoulou-Stamati et al., 2016; Trdan, 2016). Regarding the pests' management, due to the overusing of synthetic chemicals, the other side-effects such as resurgence and outbreak of new pests, several pest-resistant reports on the different classes of synthetic pesticides, and detrimental effects on valuable noun-target organisms including parasitoids and predators have also been documented (Kohler et al., 2013; Cruz et al., 2017; Sudo et al., 2018). Therefore, urgent efficacious tools for the reduction of synthetic chemical utilization and for announcing eco-friendly agents with fewer public health risks are required. Because of the low toxicity to the mammals and pose a minimum risk, the plant essential oils considered safe (Viciolle et al., 2012). The prospective pesticidal activity of several plants essential oils have been stated in recent years (Isman & Grieneisen, 2014), and the range of these eco-friendly bio-agents was extended in the present study through the introduction of Iranian phenyl- propanoid-rich summer savory as a toxic agent against two damaging coleopteran insect pests R. dominica and T. castaneum. However, based on the short residual lifetime (Isman, 2006), it is recommended that such essential oils be tested in the better applicable form such as "controlled release technique" through micro- and nano-encapsulation. 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