Investigation of the toxicity of ethanol extracts obtained from six different Satureja L. species on Colorado Potato Beetle, Leptinotarsa decemlineata (Say, 1824), (Coleoptera: Chrysomelidae)

In the present study, ethanol extracts obtained from Satureja cilicica P. H. Davis, Satureja cuneifolia Ten, Satureja hortensis L., Satureja spicigera (C. Koch) Boiss., Satureja thymbra L. and Satureja montana L. were tested against on the adults and larvae of Colorado potato beetle (Leptinotarsa decemlineata (Say, 1824)). The experiments were conducted in glass Petri dishes and vacuum desiccators including 15 individual for each period with three replicates under laboratory conditions. 10, 15 and 20 mg/mL doses of ethanol extracts conducted in the Petri dishes and the desiccators showed that depending on concentration increase and duration of exposure time resulted between 2.22-100% toxic effects on the potato beetle larvae and adults. In Petri trials, the highest mortality rate was recorded as 100% for the second larval stage at the 20 mg/mL dose of S. spicigera ethanol extract after 96 hours the treatment. In desiccator experiments, the highest toxicity rate was determined as 100% for first larval stage at the 20 mg/mL dose of S. thymbra ethanol extract after 96 hours of the application. In addition to, when LD values of the ethanol extracts were taken into account the highest toxicity of adult period was determined for S. thymbra extract (LD25: 0.000, LD50: 0.010 μL/insect), the lowest toxicity was determined for S. cilicica extract (LD90: 436.020 μL/insect). The results obtained from this study suggested that the ethanol extracts of tested Satureja L. species could be used for L. decemlineata larvae and adults as bio-larvicides and insecticides.


Introduction
The Colorado potato beetle, Leptinotarsa decemlineata (Say, 1824), (Coleoptera: Chrysomelide) is the most destructive pest in the potato cultivations and damages on many plants (such as eggplants, some tomato species) of the Solanaceae family (Popova, 2014;Alkan et al., 2015). Both adults and larvae feed on the greens of the host plants. However, larval stages are the most damaging life process that causes economic harm (Ferro et al., 1983). In studies conducted, it was determined that the pest resulted in loss of 70% -80% of potatoes (Oerke et al., 1994). Many synthetic chemicals are broadly used to control this pest. However, these synthetic pesticides can cause environmental, soil and water pollutions in the environment (Barnard et al., 1997;Gelman et al., 2001). But, due to the threat posed to the natural environment and the fact of pest vaccination to the active substances contained in these compounds (Szendrei et al., 2012) it is important to try to find non-chemical methods of controlling the pest. So, there is an increasing interest in new alternative biopesticides, insect growth regulators, natural products such as plant essential oil and extracts and secondary metabolites for pest control in agricultural production by many researchers (Hoffmann and Frodsham 1993;Gonzalez-Coloma et al., 1995, 1998Hu et al., 1999;Isman 2000;Chiasson et al., 2001;Zolotar et al., 2002;Scott et al., 2003Scott et al., , 2004. These metabolite products have been tested against many insect pest species and hopeful results for control of L. decemlineata have been reported (Hough-Goldstein, 1990;Scott et al., 2003Scott et al., , 2004Gokce et al., 2006;Alkan et al., 2015;Tampe et al., 2015). Therefore, the number of studies on plant extracts and oils has been increasing rapidly in recent years in the world (Gokturk et al., 2017;Duru et al., 2003;Kordali et al., 2007aKordali et al., , 2007b2008;2009).
The genus Satureja L. (savory), which is one of the most important genera belonging to Lamiacaeae family in Turkey and throughout the world. These families are reported nearly 7.000 species belonging to more than 230 genera (Zarshenas and Krenn, 2015). Among those genera, Satureja (savory) includes over 200 different herbs and shrubs, often aromatic, widely distributed in the Mediterranean area, Asia (Cronquist, 1988). In Turkey, there are 40 Satureja species (42 taxa) and 18 of them are endemic (Öztekin, 2012). Satureja species are known as "kekik", "sivri kekik", "kılıç kekik", "keklik otu", "catlı" or "firubi" by their names among local people in Anatolia (Başer et al., 2001). The leaves, flowers and stems of Satureja species are used as herbal tea, and also to treat infectious diseases in traditional medicine (Güllüce et al., 2003). Satureja species is high rated essential oil containing and the yield of essential oil often changes to 5% in different species of this genus (Momtaz et al., 2010). Satureja essential oils contain main monoterpenes such as "carvacrol" and "thymol". (Hadian et al., 2010). Essential oils and extracts of this genus have shown antibacterial, fungicidal, antiviral and insecticidal activities. So, they can be used as natural pesticides (Michaelakis et al., 2007). Insecticidal impact experiments of different essential oils, extracts and some monoterpen components have been broadly studied against various insects by many researchers (Lee et al., 2003;Kordali et al., 2007a;Bashır et al., 2013).
The main aim of this study was to determine the toxic effects of ethanol extracts obtained from six Satureja species against the 1 st , 2 nd , 3 rd and 4 th instars larvae and adults of L. decemlineata Petri dishes and desiccator in laboratory conditions.

Plant materials and extraction
The Collected plant materials were dried in a shady room and powdered by grinding in the grinder (about 0.100-0.400 mm particle). Then, 100 g of each sample was individually extracted with ethanol (400 mL×6) at room temperature. The extracts were filtered using Whatman filter paper (No. 1) and then concentrated under reduced pressure at 40ºC using a rotary evaporator (RV 05 Basic 1B IKA Group, Wilmington, NC, U.S.A.). Residues of each plant species were diluted with sufficient HPLC grade ethanol (Sigma-Aldrich, Milwaukee, WI, U.S.A.) and sterile water to give 100% (w/w) stock solutions. The extracts (yields 11, 7.6, 8.8, 16.2, 8.06 and 17% respectively) were stored in a freezer at 4ºC until further tests.

Bioassays using ethanol extracts
Glass Petri dishes (9 cm wide×1.5 cm deep, corresponding to 120 ml volume) were used as exposure chambers to test the toxicity of ethanol extracts of six plants against adults and larvae of Leptinotarsa decemlineata (Say, 1824). The ethanol extracts were dissolved in Ethanol-water solution (10%, v/v) to determine their contact toxicity effects. The final concentrations of the treatments were 10, 15 and 20 mg/mL.
A filter paper was placed in bottom of each of the Petri dishes (9 cm×1.5 cm deep). Then, 15 adults and larvae of L. decemlineata were placed on this filter paper, containing the appropriate amounts of potato leaves. Thus, there was direct contact between the extracts and the adults and larvae. The emulsions were sprayed to Petri dishes (9 cm diameter) and two layers of filter paper were placed in the bottom (1 ml/Petri dish). 10, 15 and 20 mg/mL doses of the ethanol extracts were sprayed to adults insects by using spray equipment. The Petri dishes were covered with a lid and transferred into incubator, and then kept under standard conditions of 25 ± 1 • C, 64 ± 5 relative humidity and 16:8 (light: dark) photoperiod for 4 days. The toxic effects against adults and larvae were tested using 20 mg/mL dose of ethanol extracts in the desiccator test. In this method, 5 liters of vacuum desiccators 250 mm in diameter disinfected with 1 % sodium hypochlorite were placed in 15 larvae and adult individuals of each potato beetle period. Inside the desiccator, 10 mL of standard glass tubing was added to 1/3 of pure water, and potato plant branches were placed in the tubes. Doses of 20 mg/mL of ethanol extracts diluted in the solvent-water solution were sprayed at a rate of 2 ml per desiccator and to thoroughly soak the potato leaves.
The treatments were arranged in a completely randomized design with three replications including controls. Izoldesis 2.5 EC (Deltametrin) (10, 15 and 20 mg/mL) was used as positive control in the same above mentioned conditions. After exposure, the mortality of the adults was counted at 24, 48, 72 and 96 h. Sterile water and Ethanol were used as control in the same way. Each experiment was replicated three times at each dose.

Biological material
The adults and larvae of Leptinotarsa decemlineata were collected from potato fields (Tepe and Söğütlü villages) at Eastern Anatolia (Erzurum) in Turkey and were reared in laboratory at 25±1 o C, 64±5 relative humidity in the Department of Plant Protection at Atatürk University. First, second, third and fourth instar larvae (determined according to their head length and width of the body) and 3-5 day-old adults were used as test insects and larvae. The cultivation of potato plants was grown in 25 square meter area belonging to Department of Plant Protection, in Agriculture Faculty, at Atatürk University and the tested insects and larvae feed on fresh leaves provided from this field. All tests were carried out under the same laboratory conditions.

Data Analysis
The results of mean mortality were subjected to one-way variance analyses (ANOVA), using SPSS 17.0 software package. Differences between means were tested through Duncan's test was used for comparison between means. Significance of differences between means was determined at p < 0.05. LD 25 , LD 50 and LD 90 values were calculated according to the method of Finney (1971). Probit analysis of concentration-mortality data was conducted to estimate the LD 25,50,90 values and associated 95 % confidence limits for each treatment (EPA Probit Analysis).

Insecticidal activity extracts
The insecticidal and larvicidal effects of ethanol extracts of Satureja cilicia, S. cuneifolia, S. hortensis, S. spicigera, S. thymbra and S. montana were studied on the 1 st , 2 nd , 3 rd and 4 th instars larvae and adults of the L. decemlineata. Petri dish and desiccator in laboratory conditions at different concentrations and exposure times were investigated. Maximum mortalities were recorded after 96 h of exposure at all concentrations (Table 1, 2, 3, 4, 5, 6 and 7). The results showed that ethanol extracts of S. cilicia, S. cuneifolia, S. hortensis, S. spicigera, S. thymbra and S. montana had significant toxic effects on both the larvae and adults of L. decemlineata comparison with the negative control and positive control (Izoldesis). In the larvae and adults the mortality increased with increasing doses of the ethanol extracts and exposure time. Varience analysis showed that the effects of ethanol extracts extracted from six different Satureja species on the mortality rates among 1 st , 2 nd , 3 rd and 4 th instars larvae and adults of L. decemlineata were highly significant on the foundation of concentration and exposure time tested (Table 1, 2, 3, 4, 5, 6 and 7). The lowest mortality rates were recorded at the different exposure time (12, 24, 48 and 72 hrs) and in the same dose (10 mg/mL) of S. cuneifolia ethanol extract (8.88, 22.2, 42.2 and 60.0%) on the 1 st instar larvae of L. decemlineata (Table 1).
Besides, the lowest mortality rates (77.7%) was found at the 96 hrs of treatment with the 10 mg/mL dose for S. cilicica and S. hortensis ethanol extracts on the 1 st instar larvae of L. decemlineata. But, the highest mortality rates (31.1% after 12 h of ethanol extracts of S. thymbra and S. montana); (48.8% after 24 hrs of ethanol extracts of S. cuneifolia and S. spicigera); (66.6% after 48 hrs of ethanol extract of S. spicigera); (80.0% after 72 h and 95.5% after 96 hrs of ethanol extracts of S. spicigera and S. thymbra) of treatment in the 20 mg/mL dose of ethanol extracts on the 1 st instar larvae of L. decemlineata. After 96 h of the treatment, the lowest mortality rate (88.8%) was recorded in the 10 mg/mL dose of S. cilicia ethanol extract, while, the highest mortality rate (100%) was in the 10 and 20 mg/mL doses of S. thymbra ethanol extract and in the 20 mg/mL dose ethanol extracts of S. spicigera and S. montana on the 1 st instar larvae of L. decemlineata. However, the mortality rates of izoldesis using as positive control were established as 95.5, 97.7 and 100% after 12 h in the 10, 15 and 20 mg/mL doses for 1 st instar larvae of L. decemlineata, respectively. Additionally, the mortality rates after 24, 48, 72 and 96 hrs of treatment with all doses (10, 15 and mg/mL) of izoldesis were found as 100% for 1 st instar larvae of L. decemlineata. No mortality for 1 st instar larvae of L. decemlineata (except 0.0% 12h; 2.22% 24 h; 4.44% 48 h;6.66% 72 h and 96 h) in the negative control. The lowest mortality rate was recorded at the different exposure time (12, 24, 48,72 and 96 hrs) and in the dose (10 mg/mL) of S. cilicica ethanol extract (4. 44, 17.7, 28.8, 46.6 and 66.6%) but, after 96 h of the treatment, the highest mortality rate (100%) was found of in the 20 mg/mL dose of ethanol extract of S. spicigera on the 2 nd instar larvae of L. decemlineata. Additionally, the mortality rates after both at all times and at all doses of izoldesis were found as 80.0-100% for 2 nd instar larvae of L. decemlineata. No mortality for larvae (except 0.0% 12h; 2.22% 24 h; 4.44% 48 h;6.66% 72 h and 96 h) in the negative control (Table 2). In comparison with the mortalities of six Satureja species ethanol extracts, the lowest mortality rates were recorded between 6.66% and 73.3 % in all doses and all times on the 3 rd instar larvae of L. decemlineata. Likewise, the highest mortality rates were found between 24.4 and 91.1% 3 rd larvae and the mortality rates after 12, 24, 48, 72 and 96 h of treatment with all doses of izoldesis were found from 91.1 to 100% for 3 rd instar larvae of L. decemlineata. No mortality was for larvae (except for 0.0% 12 h; 2.22% 24 h; 4.44% 48 h and 72; 6.66% 96 h) in the negative control (Table 3). Similarly, the lowest mortality rates were recorded at the different exposure time and in the same dose (10 mg/mL) test of ethanol extracts between 2.22% and 66.6% on the 4 th instar larvae of L. decemlineata. However, after 96 h of treatment, the highest mortality rates were determined in the 20 mg/mL concentration of S. montana ethanol extract as 93.3% for 4 th larvae. Besides, the mortality rates both at all times and at all doses of izoldesis were found between 95.5 and 100% for 4 th larvae and no mortality for 4 th instar larvae of L. decemlineata (except 0.0% 12h; 2.22% 24 h, 48 h, 72 h and 96 h) in the negative control (Table 4). When looking at adults, the lowest mortality rates were showed as 2.22% at 12 h, 13.3% at 24 h, 31.1% at 48 h, 51.1% at 72 h and 71.1% at 96 h in the 10 mg/mL for S. thymbra ethanol extract. However, the highest toxicity rates after 96 h treatment final concentration 20 mg/mL of S. spicigera and S. montana ethanol extracts were calculated as 86.6% on adults (Table 5). In addition, the mortality rates after both at all times and at all doses of Izoldesis were estimated between 93.3 and 100% against the adults of L. decemlineata. But, there was no mortality adults in the negative control groups during the test period. (Table 5).
The LD 25 , LD 50 and LD 90 values after 96 h were estimated for 1 st , 2 nd , 3 rd and 4 th instars larvae and adults of the L. decemlineata. According to LD values, although the lowest toxic effects (LD 90 ) were found 436.020 mg/mL for S. cilicica ethanol extract, again the most toxicity effects were determined as 0.000 and 0.010 mg/Petri (LD 25 and LD 50 ) for S. thymbra ethanol extracts on the adults of L. decemlineata, respectively (Table 6).
In the desiccator experiments, the maximum toxicity rates were found in higher concentration and longer exposure times on 1 st , 2 nd , 3 rd and 4 th instar larvae and adults of the L. decemlineata when compared with controls. The analysis results showed that the lowest mortality rates were observed as 11.1% after 12 h, 28.8% 24 h, 44.4% 48 h, 62.2% 72 h and 80.0% 96 h in the 20 mg/mL dose of S. cilicica ethanol extract on the 1 st instar larvae of L.  decemlineata. But, the highest mortality rates were estimated as 100% after 96 h of treatment same concentration of S. thymbra ethanol extract on the larvae. In addition, the mortality rates after different times and 20 mg/mL of concentration of Izoldesis were recorded between 97.7 and 100% on the larvae (Table 7). Similarly, the lowest mortality rates were showed between 11.1 and 80.0%, while the highest mortality rates after 12 h 22.2%, 24 h 46.6%, 48 h 66%, 72 h 88.8% and 96 h %93.3 in the 20 mg/Petri for S. thymbra ethanol extract on the 2 nd instar larvae of L. decemlineata. The mortality rates after different times and 20 mg/mL of concentration of Izoldesis were determined 100% on the larvae (Table 7). In the 3 rd instar larvae of L. decemlineata, the lowest mortality rate in the 20 mg/mL dose after 96 h of treatment was reckoned as 75.5% for S. cilicica ethanol extract. However, the highest mortality rate at the same exposure time and in the same dose was 95.5% for S. hortensis ethanol extract. The mortality rates at all times of Izoldesis used as positive control were determined as 97.7-100% for the 3 rd instar larvae (Table 7). Similarly, the lowest mortality rates were between 11.1% and 80.0%, while the highest mortality rates after 72 h 82.2% and 96 h %91.1 in the 20 mg/mL were found for S. spicigera ethanol extract on the 4 th instar larvae of L. decemlineata ( Table 7). The mortality rates at different times and in the 20 mg/mL concentration of Izoldesis were found between 97,7% and 100% larvae (Table 7). Additionally, the lowest mortality rates were recorded between 11.1% and 77.7%, while the highest mortality rates were determined after 96 h %95.5 in the 20 mg/mL for S. thymbra ethanol extract on the adults of L. decemlineata. The mortality rates at different times and in the 20 mg/Petri concentration of Izoldesis were found between 93.3% and 100% for L. decemlineata adults. But, there was no mortality in the 1 st , 2 nd , 3 rd and 4 th instar larvae and L. decemlineata adults in the negative control groups during the test period (Table 7).
Toxic effects of plant extracts, essential oils and various secondary metabolite products have been reported in different researches (Kesdek et al., 2015;Usanmaz et al., 2016;Kısa et al., 2018). The present study showed that under in vivo (between 2.22 and 100%) and in vitro (between 8.88 and 100%) conditions, the ethanol extracts of six Satureja plant species had the strong insecticidal activity based on the mortality of all the tested (1 st , 2 nd , 3 rd and 4 th ) instars larvae and adults of L. decemlineata. (Table 1, 2, 3, 4, 5, 6 and 7). The results are in agreement with the previous literature reports on plant extracts (Kesdek et al., 2014;Güzel et al., 2017). The successful result was obtained from the ethanol extracts. It was demonstrated that the wild thyme (Thymus serpyllum L.) water extracts had toxic effects at different concentrations on 4 th instars larvae and adults of L. decemlineata (Rusin et al., 2016). In this study, we have found that six Satureja species ethanol extracts have a toxic effect (between 2.22 and 93.3%) in the 10, 15 and 20 mg/Petri concentrations on adults and 4 th instar larvae of L. decemlineata (Table  5). In a previous study, it was found that the ethanol extracts of M. chamomilla had toxic effects on the L 3 and L 4 larvae (44.83% and 42.87%) of L. decemlineata (Biniaś et al., 2017). Besides, it was reported that ethanol extracts of five Vincetoxicum species had toxicity in the different doses and at exposure times on 3 rd instar larvae of L. decemlineata (Güzel et al., 2017).
In the current study, we have found that the ethanol extracts of Satureja species have larvicidal effects in all the exposure times (12, 24, 48, 72 and 96 hrs) and treatment doses (10, 15 and 20 mg/mL) with mortality rates (between 2.22% and 100%) on the 1 st , 2 nd ,3 rd and 4 th instar larvae of L. decemlineata (Table 1, 2, 3 and 4). Previous studies showed that the extracts obtained from S. officinalis and R. officinalis plant species had insecticidal effects between 85.9 and 97.5% mortality rates under field and laboratory conditions on adults of L. decemlineata (Kara et al., 2014). In our desiccator work, it was determined that the ethanol extracts obtained from six Satureja species had important insecticidal effects (with between 2.22% and 93.3% the mortality rates) in all exposure times and treatment dose (20 mg/mL) on L. decemlineata adults (Table 7).
Many studies conducted with desiccator trials; Topuz et al. (2018) presented M. pulegium essential oil to be the most toxic oil against Tetranychus urticae in all the biological stages tested (LC 50 = 0.60 μL/L air for eggs, 0.60 μL/L air for larvae and 0.49 μL/L air for adult females), followed by F. vulgare essential oil (LC 50 = 2.67 μL/L air for eggs and adult females, and 2.56 μL/L air for larvae). In the same way, it was stated that the essential oils of three different plant species had a strong insecticidal activity under desiccator conditions on Tribolium confusum and Sitophilus granarius adults (Yıldırım et al., 2005). In another study, it was determined that the extracts obtained from three different plant species were effective against L. decemlineata larvae (Pavela, 2010). In our study, we found that the ethanol extracts obtained from six Satureja species have larvicidal effects all the exposure times and treatment (20 mg/mL) between 8.88% and 100% with the mortality rates on the 1 st , 2 nd , 3 rd and 4 th instars larvae and adults of the L. decemlineata (Table 7). Emsen et al., (2012) reported that two lichen extracts had an important insecticidal effect on 4 th instar larvae and adults of L. decemlineata. The same researchers stated that the most efficient crude extracts on the 4 th instar larvae and adults of L. decemlineata was diffractaic acid (LC 50 = 1.509 and 1.783 ppm, respectively). In the present study, we have determined that the most effective ethanol extract on the 4 th instar larvae and adults of L. decemlineata was for S. thymbra plant (LD 50 =2.127 and 0.010 ppm, respectively) ( Table 7).
On the other hand, in our study, we recorded that the most toxicity effects of S. spicigera (in the LD 50 value) and S. thymbra ethanol extracts (in the LD 90 value) were 0.873 and 10.350 on the 1 st instar larvae L. decemlineata, respectively. At the same time, it was determined that the ethanol extracts of S. montana were 0.205 in the LD 50 and 1.016 in the LD 90 values on the 2 nd instar larvae of L. decemlineata. In addition, it was stated that the highest toxicity effects of S. cuneifolia ethanol extracts were found as 0.312 in the LD 50 and 19.241 in the LD 90 values on the 3 rd instar larvae of L. decemlineata (Table 7).    According to this information, it can be suggested that these tested plant extracts contain the high content of these compounds and can be used as new insecticidal test subjects against L. decemlineata.

Conclusion
As a result, the development of biological insecticides will help to reduce the adverse effects on environmental of synthetic chemicals. In the present study, ethanol extracts obtained from Satureja cilicia, S. cuneifolia, S. hortensis, S. spicigera, S. thymbra and S. montana plant species had the toxic effects on the 1 st , 2 nd , 3 rd and 4 th instar larvae and adults of L. decemlineata. In this respect, it can be suggested that the ethanol extracts obtained from these Satureja species can be noted as potential bio-insecticides alternatives to control against the all the instar larvae and adults of L. decemlineata in agricultural pruducts. But, further studies are necessary to determine whether it could have value in the struggle of L.decemlineata.