Plant extracts can be used as an alternative to synthetic insecticides for the control of insect pests. Based on this knowledge, juvenomimetic and insecticidal activities of n-hexane extracts of the aerial parts of Senecio salignus DC. (Asteraceae) and Salvia microphylla Kunth (Lamiaceae) collected in Mexico were evaluated against 1st instar larvae of Spodoptera frugiperda Smith & Abbot (Lepidoptera: Noctuidae). Senecio salignus extract showed insecticidal activity at 500 ppm, resulting in larval mortality of 52.5% and pupal mortality of 62.5%. Salvia microphylla extract at the same concentration caused larval mortality of 65.0% and pupal mortality of 82.5%. The LC50 was 440 ppm for S. salignus extract and 456 ppm for S. microphylla extract based on the total larval period. The juvenomimetic activity of S. salignus extract at 500 ppm increased the duration of the larval period to 17.3 d and of the pupal period to 1.4 d. It also reduced pupal weight by 34.7% with respect to the control (241 mg). For S. microphylla extract at 500 ppm, the duration of the larval and pupal periods were increased by 2.0 and 12.1 d, respectively, and the pupal weight was reduced by 14.1% with respect to the control (243 mg). The major compounds of S. salignus extract were γ-sitosterol, palmitic acid, lupeol, and β-amyrin, and those of S. microphylla extract were oleic acid, γ-sitosterol, (Z,Z,Z)-9,12,15-octadecatrien-1-ol, and palmitic acid. These results indicate that both extracts have potential to be used to control S. frugiperda due to their juvenomimetic and insecticidal activities.
The fall armyworm, Spodoptera frugiperda Smith & Abbot (Lepidoptera: Noctuidae), is one of the most destructive insect pests of maize, Zea mays L. (Poaceae), in the tropical and subtropical regions of the western hemisphere (Andrews 1988; Santos et al. 2003). Currently, the principal control method for this species is through the use of synthetic insecticides (Tagliari et al. 2010). However, integrated pest man agement programs for noctuid insects have been well demonstrated. As a result, there has been increased interest in research related to the identification of botanical extracts that demonstrate insecticidal activity, so as to reduce the use of synthetic insecticides (Pavela & Chermenskaya 2004).
Many plants have insecticidal or juvenomimetic activities against insects. The genus Senecio (Asteraceae) comprises about 1,500 species with 165 species found in Mexico. These species are known to produce many insecticidal compounds such as alkaloids, sesquiterpenes, chalcones, and flavonoids (Romo de Vivar et al. 2007). This genus has also been associated with anti-inflammatory, vasodilator, antiemetic, and antimicrobial activities (Rodríguez & López 2001; Rosa et al. 2004). Studies of insecticidal activity have been conducted with Senecio umbrosus Waldst. & Kit and Senecio otites Kunze ex DC., and extracts have been shown to affect larvae of Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) (Domínguez et al. 2008; Pavela 2011).
The genus Salvia is the most diverse genus of the family Lamiaceae, with over 1,000 species around the world distributed in tropical and subtropical zones. In Mexico, there are at least 300 reported species (Fernández 2006). Many species of this genus produce various bioactive compounds such as sesquiterpenes, diterpenes, triterpenes, sterols, and polyphenols (Yi-Bing et al. 2012). Antioxidant, antimicrobial, analgesic, anticancer, antipyretic, and anti-inflammatory activities have also been reported (Kamatou et al. 2008; Akin et al. 2010). In addition, insecticidal activity on S. frugiperda and S. littoralis has been reported (Pavela 2004; Zavala-Sánchez et al. 2013).
The aim of this study was to determine the insecticidal and juvenomimetic activities of the n-hexane extracts of the aerial parts of “chilca,” Senecio salignus DC. (Asteraceae), and “mirto,” Salvia microphylla Kunth (Lamiaceae), on S. frugiperda.
Materials and Methods
Fall armyworm larvae were reared in the Laboratory of Natural Insecticide Compounds at the Universidad Autónoma de Querétaro, in Querétaro, Mexico. The larvae were reared at 25 ± 2 °C and 70% relative humidity with a 12:12 h L:D photoperiod. For preparation of the fall armyworm diet, the following mixture was made: 800 mL distilled water, 30 g ground beans, 90 g ground corn (dried beans and corn were ground with a Thomas-Wiley Model 4 mill with a particle size of 1 mm), 20 g brewer's yeast, 10 g vitamins (vitamin mix, Lepidoptera # 722, Bio-Serv), 10 g agar, 1.7 g ascorbic acid (dissolved in 17 mL ethanol), 2.5 mL formaldehyde, 1.7 g methyl p-hydroxybenzoate, and 0.6 g neomycin sulfate (Bergvinson & Kumar 1997).
PLANT MATERIAL AND EXTRACTION
Aerial parts (leaves, stems, and flowers) of S. salignus and S. microphylla were collected in Tenancingo County, Mexico, at 18.9514° to 19.0403° north latitude and 98.5958° to 99.6436° west longitude, and 2,060 m asl. in Sep 2013. Taxonomic authentication was performed by Abigail Aguilar Contreras, and vouchers were deposited at Herbarium of Instituto Mexicano del Seguro Social (IMSS). The voucher specimen for S. salignus was IMSS M 15,546 and for S. microphylla was IMSS M 15,821.
Phytochemical test of the n-hexane extracts of Senecio salignus and Salvia microphylla.
After thorough cleaning of aerial material from each plant, it was shade dried at room temperature for a minimum of 20 d. The dried plant was then made into powder with a Thomas-Wiley Model 4 mill with a particle size of 1 mm. Dried and powdered aerial material from S. salignus or S. microphylla (250 g) were extracted with 2 L n-hexane under reflux for 4 h. The extract was filtered and the solvent removed under reduced pressure by using a rotatory evaporator. The yield weight of S. salignus extract was 2.31% and of S. microphylla 2.09%.
The extracts were tested in triplicate for various phytochemical classes by using the following methods: 1) alkaloids: Meyer, Wagner, and Dragendorff reagents; 2) cardiotonics: Raymond and Baljet reagents; 3) flavonoids: H2SO4 concentrate; 4) saponins: H2O at boiling temperature; 5) sterols and triterpenes: Salkowsky reagent, Liebermann/ Burchard reagent; 6) tannins: ferric chloride; and 7) terpenes: Noller reagent (Tiwari et al. 2011; Wadood et al. 2013).
IDENTIFICATION OF THE PRINCIPAL COMPOUNDS FROM NHEXANE EXTRACTS OF S. SALIGNUS AND S. MICROPHYLLA
Twenty µL n-hexane extracts of aerial portions of S. salignus and S. microphylla were diluted with 1 mL acetone. The extracts were analyzed on an Agilent Technologies (Santa Clara, California) 6890N GC equipped with an HP-5MS column (30 m in length; 25 mm internal diameter; 0.25 µm film thickness) equipped with an Agilent MS 5973 detector, at 250 °C. The carrier gas was helium, with a flow rate of 1 mL/min; the split ratio was 2:1. The column temperature was initially 50 °C (for 3 min) and was gradually increased to 240 °C, at 3 °C/min; this temperature was maintained for 2 min. The injector temperature was 250 °C, and 1 µL n-hexane extract was injected and analyzed in duplicate. The spectra were collected at 71 eV ionization voltages, and the analyzed mass range was 15 to 600 m/z. The identification of the components was confirmed by comparison of the retention indices with those of authentic compounds and with the Wiley09/ NIST11 library.
Bioassays were conducted using for each concentration 40 replicates (larvae) divided in 5 experimental units with 8 larvae each, selected randomly. Preliminary screening of each extract was carried out at 5 concentrations ranging from 0.5 to 5,000 ppm, and a control, by following the previously described method (Santiago-Santiago et al. 2009), altogether using 240 larvae for each plant. The extracts were mixed with the larval diet ingredients during preparation. For the final bioassays, 6 concentrations of extracts were tested (0, 50, 500, 1,000, 2,000, and 5,000 ppm) according to the method used by Rodríguez-Hernández & Vendramim (1996) as modified by Romero-Origel et al. (2012), and 240 larvae were used for each plant. The larvae were maintained at 27 ± 2 °C, 70 ± 5% relative humidity, and a 14:10 h L:D photoperiod. The pupae were weighed 24 h after pupation and then moved to another container for development to the adult stage. The insecticide parameters evaluated were larval and pupal mortality, and the juvenomimetic parameters were the length of the larval and pupal period and the pupal weight at 24 h after formation. The median lethal concentration (LC50) to the larval population of S. frugiperda was calculated for each extract by using data for total larval period mortality.
Insecticidal and juvenomimetic activities of aerial parts n-hexane extract of Senecio salignus against Spodoptera frugiperda.
A statistical analysis was conducted, and data were tested for normality and homoscedasticity before analysis. In some cases, Kruskal—Wallis non-parametric analysis of variance (ANOVA) was used when data violated these assumptions and could not be corrected using a transformation. ANOVA, followed by Tukey's test, was performed, and the LC50 was calculated by probit analysis, using the SYSTAT statistical analysis program (SYSTAT 1998).
The extract of S. salignus tested positive for tannins, flavonoids, terpenes, triterpenes, and sterols (Table 1). The S. microphylla extract tested positive for cardiotonics, saponins, tannins, terpenes, triterpenes, and sterols (Table 1).
Insecticidal and juvenomimetic activities of aerial plant tissue n-hexane extract of Salvia microphylla against Spodoptera frugiperda.
INSECTICIDAL ACTIVITY OF S. SALIGNUS AND S. MICROPHYLLA EXTRACTS
Exposure to the n-hexane extract of the aerial parts of S. salignus (Table 2) induced 100% larval mortality at 5,000 ppm, and 95, 90, and 52.5% at 2,000, 1,000, and 500 ppm, respectively. Mortality was 10% in the control treatment (LC50 = 440 ppm). Pupal mortality was 100, 97.5, 95, and 62.5% at 5,000, 2,000, 1,000, and 500 ppm, respectively, and the control mortality was 15%. The S. microphylla extract (Table 3) resulted in a larval mortality of 100% at 5,000 ppm and 97.5, 87.5, and 65% at 2,000, 1,000, and 500 ppm, respectively. The control showed 7.5% mortality (LC50 = 456 ppm). Pupal mortality was 100, 100, 95, and 82.5% at 5,000, 2,000, 1,000, and 500 ppm, respectively, and 15% in the control.
JUVENOMIMETIC ACTIVITY OF S. SALIGNUS AND S. MICROPHYLLA EXTRACTS
The juvenomimetic activity of the S. salignus n-hexane extract (Table 2) extended the larval period by 31.6, 29.1, and 17.3 d at 2,000, 1,000, and 500 ppm, respectively, and increased the pupal period by 8.9, 5.9, and 1.4 d at 2,000, 1,000, and 500 ppm, respectively, compared with the controls (22.9 and 11.1 d). It also reduced the pupal weight by 63.7, 54.4, and 34.7% at 2,000, 1,000, and 500 ppm when compared with the control weight (241 mg).
The S. microphylla extract (Table 3) prolonged the larval period by 12.5, 10.9, and 2.0 d at 2,000, 1,000, and 500 ppm, respectively, and increased the pupal period by 16.5 and 12.1 d at 1,000 and 500 ppm, respectively, compared with controls (22.5 and 10.5 d). It also reduced the pupal weight by 74.9, 39.2, and 14.1% at 2,000, 1,000, and 500 ppm, respectively, when compared with the control (243 mg).
IDENTIFICATION OF PRINCIPAL COMPOUNDS
We found 48 compounds in n-hexane aerial extracts of S. salignus identified by GC-MS analysis, representing 99.95% of the extracted material (Table 4); the retention times ranged between 3.88 and 67.20 min. The major components and their respective retention times were: palmitic acid (12.23%) 44.66 min, γ-sitosterol (16.10%) 54.05 min, β-amyrin (5.18%) 61.51 min, and lupeol (6.44%) 61.72 min. The GCMS analysis of n-hexane aerial parts extracts of S. microphylla showed 59 compounds, which accounted for 99.96% of the extracted material (Table 5); the retention times ranged between 3.72 and 63.39 min. The major components and retention times were: palmitic acid (7.12%) 44.8 min, (Z,Z,Z)-9,12,15-octadecatrien-1-ol (11.11%) 49.76 min, oleic acid (14.76%) 49.98 min, and γ-sitosterol (12.77%) 54.74 min.
Composition of the n-hexane aerial plant tissue extract of Senecio salignus.
To our knowledge, this is the first report to demonstrate the insecticidal and juvenomimetic activities of the n-hexane extracts of aerial parts of S. salignus and S. microphylla against S. frugiperda larvae. These extracts demonstrated strong insecticidal activity and showed an LC50 of 440 ppm and 456 ppm, respectively. In similar studies, 0.5% of root powder of S. salignus caused 100% mortality in Zabrotes subfasciatus Boheman (Coleoptera: Bruchidae) in stored beans (López-Pérez et al. 2007; López et al. 2010). Moreover, the extracts of Lepidaploa lilacina Mart. ex DC. (Asteraceae), Ageratum fastigiatum Gardner (Asteraceae), and Lychnophora ramosissima Gardner (Asteraceae) caused 72.0, 65.9, and 61.0% egg mortality, respectively (Rodríguez & López 2001). Rodríguez & López (2001) also showed that after 2 d, Lychnophora sp. (Asteraceae) and Vernonia holosericea Mart. (Asteraceae) extracts caused 8.7 and 87% larval mortality, respectively, in Z. subfasciatus. The extracts of Lychnophora ericoides Mart. (Asteraceae) and Trichogonia villosa Sch. Bip. ex Baker (Asteraceae) caused 97.7% egg mortality in S. frugiperda after 1 d (Tavarez et al. 2009).
Composition of the n-hexane aerial plant tissue extract of Salvia microphylla.
Additionally, the chloroform extract of aerial parts of S. microphylla showed insecticide activity against S. frugiperda (LC50 = 919 ppm) (Zavala-Sánchez et al. 2013). On the other hand, Ramírez-Moreno et al. (2001) reported that aqueous extracts of aerial parts at 5% concentration of Salvia karwinskii Benth (Lamiaceae) and Salvia polystachya Epling (Lamiaceae) had low insecticidal activity (13% with both species) against Leptophobia aripa elodia Boisduval (Lepidoptera: Pieridae). Rashid et al. (2009) showed 80% mortality in adults of Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) with dichloromethane extract of the aerial parts of Salvia cabulica Benth (Lamiaceae).
The juvenomimetic activities of S. salignus and S. microphylla nhexane extracts against S. frugiperda larvae began at 500 ppm, wherein each extract increased the length of the larval and pupal periods and decreased the pupal weight. Ramírez-Moreno et al. (2001) tested the repellent activity of aqueous extract using the powder of the entire S. salignus plant on L. aripa elodia. However, this plant extract had no effect on this insect. Domínguez et al. (2008) showed the antifeedant activity of the ethanolic extract of aerial parts of S. otites against S. littoralis and reported a feeding inhibition of 43% at 100 µg/cm2 in Myzus persicae Sulzer (Hemiptera: Aphididae) and Rhopalosiphum padi L. (Hemiptera: Aphididae). The same study also showed that only 30% of M. persicae and 13% of R. padi settled to feed at a concentration of 50 µg/cm2. Moreover, the chloroform extract of the aerial parts of S. microphylla showed juvenomimetic activity against S. frugiperda beginning at 500 ppm, which increased the pupal duration to 2 d and reduced the pupal weight by 13.3% with respect to the control (Zavala-Sánchez et al. 2013). Ramírez-Moreno et al. (2001) observed 7% repellency with 5% aqueous extracts of S. karwinskii and S. polystachya against L. aripa elodia larvae.
The GC-MS analysis showed that the principal components of S. salignus n-hexane extract were: palmitic acid, γ-sitosterol, β-amyrin, and lupeol, in addition to caryophyllene oxide. Sánchez-Muñoz et al. (2012) reported n-hexane extracts of aerial parts of S. salignus from the Mexican state of Guerrero to contain caryophyllene oxide, whereas Pérez-González et al. (2013) reported nonacosane (10.11%), (Z,Z)-9.12-octadecadienoic acid (7.5%), squalene (5.17%), and (Z,Z,Z)-9,12,15-octadecatrienoic acid (5%) as principal components of the chloroform extract of aerial parts of S. salignus. The principal components of S. microphylla extract were: palmitic acid, (Z,Z,Z)-9,12,15-octadecatrien-1-ol, oleic acid, and γ-sitosterol, in addition to caryophyllene and caryophyllene oxide. Lima et al. (2012) found (E)-caryophyllene (15.35%), α-eudesmol (14.06%), β-eudesmol (8.74%), and γ-eudesmol (7.64%) as principal components of essential oil from aerial parts of S. microphylla.
This study showed potential for the use of S. salignus and S. microphylla n-hexane extracts against S. frugiperda larvae. Both plants contain bioactive compounds such as flavonoids, essential oils, diterpenes, and triterpenes, which can act as an antifeedants (Tomás-Barberán & Wollenweber 1990). Also, palmitic acid and oleic acid showed insecticidal and juvenomimetic acivities against S. frugiperda larvae with larval viability values of 33.3 and 48.5%, respectively, when exposed to 1,600 ppm of palmitic and oleic acid, with respective LC50 values of 989 and 1,353 ppm, respectively (Pérez-Gutiérrez et al. 2011). These acids were present as principal components of n-hexane extract of S. microphylla, and palmitic acid was extracted from S. salignus. The γ-sitosterol was reported as an active principle of acetone extract of stem bark of Vitex schliebenii Moldenke (Lamiaceae) against 3rd and 4th instar larvae of Anopheles gambiae Giles (Diptera: Culicidae) (Nyamoita et al. 2013), and this phytosterol also was a principal component of n-hexane extracts of S. salignus and S. microphylla. The β-amyrin and lupeol isolated from Inula japonica (Asteraceae) were determined to have acaricidal activity against Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae) by Duan et al. (2011). In this study, these compounds were also present in S. salignus extract. Therefore, it is possible that the presence of palmitic acid, γ-sitosterol, β-amyrin, and lupeol in S. salignus n-hexane extract and that of palmitic acid, oleic acid, and γ-sitosterol in S. microphylla n-hexane extract are responsible for insecticidal and juvenomimetic activities in the present study. These extracts could provide a botanical source of insecticides for alternative pest management of S. frugiperda.
The authors gratefully acknowledge the National Council for Science and Technology (CONACYT) for the master's degree program scholarship, the Autonomous University of Querétaro Institutional Program for Faculty Research (FOFI-UAQ) (FCQ201408), and Q. Candy Monserrat Romero Origel for helping us collect plant specimens. The authors declare no conflicts of interest, financial or otherwise.