Insect rearing

Adult maize weevils were cultured in 10-L plastic buckets containing 500 g of corn (Zea mays L.) seeds. Each plastic bucket was populated with 15 pairs of maize weevils. The buckets were stored at room temperature (27 ± 2°C), and the lids were opened daily to allow air circulation. After 4 d, the adult maize weevils were removed from the plastic buckets, leaving only the maize weevil spawns. These spawns were preserved until the adult maize weevils emerged. Subsequently, the emerging adults were reared until a consistent and sufficient number of maize weevils was obtained for conducting the insecticidal activity bioassay using P. amboinicus essential oil. For the test, 7-d-old adult maize weevils were used.

Essential oil extraction

Essential oil of P. amboinicus was extracted using a water distillation method. Three kilograms of P. amboinicus fresh leaves were cleaned and shredded into small pieces. Next, 200 g of these plant parts were placed into 2,000-ml round flasks and mixed with 1,000 ml of distilled water. This mixture was subjected to a distillation process at 100–120°C for 3 h by using essential oil distillation equipment. After distillation, the essential oil that separated floated atop the water. This layer was purified through centrifugation at 10,000 rpm for 10 min to remove any residual water. The obtained P. amboinicus essential oil was stored in amber glass bottles at 4°C until it was further analyzed and tested for insecticidal activity.

Chemical composition analysis

The extracted essential oil was analyzed following the method of Satongrod et al. (2021) with gas chromatography (model Clarus 680 gas chromatograph, PerkinElmer)-mass spectrometry. The column used was an Rtx-5MS capillary type, with a length of 30 m, a diameter of 0.32 mm, and a thickness of 1 µm. For injection, the essential oil was used in split mode (split ratio, 1:100 v/v) at a concentration of 500,000 ppm, with a volume of 1 µl. Helium gas was used as the carrier gas at a flow rate of 1.0 mm/min. The injector temperature was set at 280°C. At column state, an initial temperature of 45°C was maintained for 5 min and then the temperature was increased at a rate of 10°C/min until it reached 200°C, where it was held for 5 min. Electron impact mode mass spectrometry conditions were 70 eV, by using a quadrupole mass analyzer. The detector temperature was set at 250°C. The substances were characterized by spectral comparison using the National Institute of Standards and Technology Mass Spectral Search Program and the Chemstation Wiley Spectral Library. They were considered comparable to known substances with mass spectra that had a quality match of >80%. The chemical composition data were analyzed based on the reading of the retention time and percent peak area.

Insecticidal activity bioassay

The fumigation toxicity of P. amboinicus essential oil against maize weevil was tested using a vapor phase test in 40-ml glass vials with lids. The experimental plan followed a completely randomized design (CRD) with five replications of seven treatments at concentrations of 0, 0.5, 1, 1.5, 2, 2.5, and 3 µl/ml air. Each concentration was prepared by diluting essential oils in acetone. In each glass vial, we placed 10 g of dried corn kernels. Next, 100 µl of the essential oil solution was aspirated using a micropipette and dripped onto a filter paper (Whatman no. 1; diameter, 2 cm). The filter paper was left to evaporate for 5 min before being placed inside the glass vial lid. Next, 7-d-old maize weevil adults were released into each glass vial, with 10 pairs/vial, and the glass bottle caps were tightly closed. The number of dead maize weevils was recorded at 24, 48, 72, 96, 120, 144, and 168 h.

Statistical analyses

Percent mortality of adult maize weevils was evaluated by the formula (Nd/Nt) × 100, where Nd is the number of dead maize weevil adults and Nt is the total number of maize weevil adults used in the bioassay. If the maize weevil mortality in the control treatments fell within the range of 5–20%, mortality for each treatment was adjusted using Abbott’s formula (Abbott 1925). The median lethal concentrations (LC₅₀) were determined using probit analysis (Finney 1971). The data were analyzed using one-way analysis of variance according to the CRD design. Treatment means were compared with the least-significant difference (LSD) test (P < 0.05) with Statistix 9.0 (Analytical Software, Tallahassee, FL).

Chemical composition of P. amboinicus essential oil

Analysis of the chemical composition of the essential oil from P. amboinicus leaves revealed 23 chemical constituents (97.51%), with thymol (49.96%) identified as the main compound followed by caryophyllene (8.77%), trans-α-bergamotene (5.11%), 3-methyl-4-isopropylphenol (4.32%), γ-terpinene (4.26%), p-cymene (3.86%), caryophyllene oxide (3.04%), humulene (2.88%), 4-hydroxy-2-methylacetophenone (2.80%), 2-hydroxy-2-phenylbutyramide (2.74%), (2-oxazolidinylidene)malononitrile (2.38%), hexestrol (2.24%), terpinen-4-ol (0.94%), 1-octen-3-ol (0.91%), (1R,7S,E)-7-isopropyl-4,10-dimethylenecyclodec-5-enol (0.82%), α-bisabolene (0.43%), isoaromadendrene epoxide (0.42%), α-farnesene (0.39%), 4-carene (0.37%), α-muurolene (0.30%), 3-hexen-1-ol (0.25%), ledene oxide-(II) (0.21%), and α-myrcene (0.14%) (Table 1). These finding were consistent with previous reports on the major constituents present in P. amboinicus essential oil, such as thymol, carvacrol, 1,8-cineole, p-cymene, and terpinen-4-ol (Murthy et al. 2009, Senthilkumar and Venkatesalu 2010). Singh et al. (2002) reported thymol (93%) as the major constituent, whereas Wanna and Krasaetep (2019) reported carvacrol (40.49%) as the major constituent. The essential oil of P. amboinicus contained two major phenolic monoterpenes, namely, carvacrol and thymol, that showed insecticidal activity (Singh et al. 2002, 2014; Wanna and Krasaetep 2019; Wanna and Kwang-Ngoen 2019; Satongrod and Wanna 2020).

Table 1. Chemical compounds of essential oil from Plectranthus amboinicus leaves.

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Insecticidal activity

The fumigation toxicity (LC₅₀) of essential oil from P. amboinicus leaves on maize weevils at 24, 48, and 72 h was 1,442.39, 292.53, and 161.35 µl/ml air, respectively (Table 2). The mortality of adult maize weevils after fumigation with P. amboinicus essential oil at seven concentrations showed significant differences (P < 0.01) within 48, 72, 96, 120, 144, and 168 h (Table 3), but not at 24 h. All concentrations of P. amboinicus essential oil resulted in <50% mortality of adult maize weevils within 48 h. An essential oil concentration of 2.5 µl/ml air at 96 and 120 h induced >90% mortality of adult maize weevils, and no significant difference was found compared with the highest concentration of 3 µl/ml air. The mortality of adult maize weevils was >90% at 2 µl/ml air within 144 and 168 h, and there was no significant difference compared with concentrations of 2.5 and 3 µl/ml air, causing the mortality of the adult maize weevils to reach as high as 99–100%. The mortality of adult maize weevils increased with a higher concentration of the essential oil solution and a longer duration of exposure. The insecticidal activity of P. amboinicus essential oil at a concentration of 3 µl/ml air resulted in a maximum 100% mortality of adult maize weevils within 144 h, which was 30 times higher than the concentration reported by Wanna and Krasaetep (2019), where 0.1 µl/ml air of P. amboinicus essential oil achieved 100% efficiency for the same duration of exposure. Satongrod and Wanna (2021) reported that essential oil from P. amboinicus at a concentration of 1 µl/L air inhibited cowpea weevil spawning by 70.53% and first-generation adult spawning by 73.30%, which is consistent with the finding of Keita et al. (2000), who stated that Ocimum spp. (Lamiaceae) could be used as an alternative to synthetic insecticides against adults and inhibit the egg laying of cowpea weevil. In addition, previous reports indicate that carvacrol, thymol, caryophyllene, humulene, terpinene, cymene, and terpineol in essential oils of P. amboinicus exhibit insecticidal, antifungal, and antibacterial properties (Negahban et al. 2007, Bhatt and Negi 2012, Gonçalves et al. 2012, Satongrod and Wanna 2020). These properties may be attributed to the presence of different types and amounts of compounds in essential oils. It is well known that the variability of the chemical compositions of essential oils depends on geography, growing season, genetics, plant age, harvest time, and method of essential oil extraction (Özcan and Chalchat 2006, Ortega et al. 2011). These findings demonstrate the potential for using these natural compounds as an effective alternative to synthetic pesticides.

Table 2. Fumigation toxicity of Plectranthus amboinicus essential oil against adult maize weevils (Sitophilus zeamais).*

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Table 3. Mortality (mean ± SD) of maize weevils (Sitophilus zeamais) exposed to essential oil of Plectranthus amboinicus leaves.

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In summary, the essential oil obtained from P. amboinicus leaves in this study was comprised of 23 chemical constituents, with thymol being the most prominent. A fumigation toxicity bioassay demonstrated high effectiveness against adult maize weevils when administered at a concentration of 2 µl/ml air, resulting in substantial mortality within 48–168 h. This underscores the potential of P. amboinicus–derived essential oil as a viable insecticidal agent for managing maize weevil populations that pose significant threats to agricultural storage. Given the variability in essential oil compositions, further investigations are imperative to pinpoint the specific compound(s) responsible for its insecticidal properties, thereby ensuring consistent treatment outcomes. Furthermore, a comprehensive elucidation of an essential oil’s mechanisms of action on insects and mammals, including the roles played by minor components, is essential before contemplating its commercial development and use.