Chemical composition and insecticidal activities of essential oils against diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae)

Abstract Five Himalayan plants namely, Acorus calamus, Cedrus deodara, Aegle marmelos, Tagetes minuta and Murraya koenigii were used for the extraction of essential oils through hydrodistillation and the major volatile constituents as identified by GC and GC–MS techniques were β-asarone (91.1%), β-himachalene (45.8%), limonene (59.5%), Z-ocimene (37.9%) and α-pinene (54.2%), respectively. Essential oils were tested for their insecticidal properties against larvae of diamondback moth, Plutella xylostella (L.) (Lepidoptera: Yponomeutidae). Results showed that A. calamus was most toxic (LC50 = 0.29 mg mL−1) to P. xylostella followed by C. deodara (LC50 = 1.08 mg mL−1) and M. koenigii (LC50 = 1.93 mg mL−1) via residual toxicity bioassay. Per cent feeding deterrence index and growth inhibition was significantly higher in A. calamus (42.20 and 68.55, respectively) followed by C. deodara (35.41 and 52.47). In repellent activity studies, C. deodara showed high repellence (64.76%) followed by A. calamus (55.05%). Graphical abstract

, repellent, fumigant (Erler et al. 2006;toloza et al. 2006) and ovicidal activities (tunc et al. 2000). Due to above properties, essential oils are gaining more importance as an alternative control of insect pests and vectors (Park et al. 2003;Prajapati et al. 2005). Besides, essential oils are potentially suitable for integrated pest management programmes. Insecticidal activity of essential oils has been reported against cabbage looper, Trichoplusia ni (Jiang et al. 2012), armyworm, Pseudaletia unipuncta (akhtar et al. 2008) and stored product pests (Dales 1996). Intensive use of chemical insecticides for the control of Plutella xylostella has led to this pest developing resistance to a wide range of insecticides and caused serious damage to natural enemies (Ke et al. 1991;talekar & Shelton 1993). Due to harmful effects of synthetic pesticides to health, environment and resistance development in pests, there is a need for the development of safer and effective alternate strategies to control the pests. In continuation to our previous programme for search of bioactive compounds, in the present investigation, we have selected five aromatic plants from western Himalayan region and the essential oils were extracted to study their chemical composition and insecticidal activities against the larvae of P. xylostella. the results of the present study would be useful for the development of new and alternate management strategies for pest control.

Essential oil composition
Various components in each essential oil were characterised by GC and GC-MS analysis on a DB-5 capillary column. Fifty-one components were identified in five essential oil samples and accounted for up to 86.1-96.6% of the total oils. all the identified essential oil constituents in each oil sample are summarised in table S1.

Residual toxicity of essential oils via leaf dip method
the response of P. xylostella larvae to the oil at the initial screening concentration (10 mg mL −1 ) was found to be differed significantly (one way aNoVa; F 14, 4 = 3.45; p < 0.001). Acorus calamus, Cedrus deodara and Murraya koenigi caused 100% mortality of larvae after 72 h and were at par (table S2). the activity of different essential oils against larvae of P. xylostella in terms of lethal concentration to kill 50% of the test population relative (LC 50 ) to control values and other statistical parameters generated by linear regression analysis is summarised and presented in tables S2 and S3. among the essential oils, A. calamus was most toxic to P. xylostella with an LC 50 of 0.39 and 0.29 mg mL −1 at 48 and 72 h, respectively, followed by C. deodara (1.08 mg mL −1 ) and M. koenigii (LC 50 = 2.98 and 1.93 mg mL −1 ); the literature on the larvicidal potential of A. calamus oil and its fractions against P. xylostella is lacking. Essential oil of A. calamus showed promising larvicidal effect against larvae of Culex quinquefasciatus (LC 50 = 63.43 mg mL −1 ) and it may be due to the presence of β-asarone and cis-β-terpineol (Senthil Kumar & Venkatesalu 2012). In a similar study, Chaudhary et al. (2011) reported the C. deodara crude oil and its enriched fractions (himachalenes and atlantones) showed larvicidal activity to P. xylostella. Further, C. deodara oil shows toxicity to Lymnaea acuminata when combined with Azadirachta indica and Embelia ribes extracts (Rao & Singh 2001). In the present study, the essential oil of C. deodara showed larvicidal which may be due to the presence of major compounds (β, α and γ himachalene).

Repellent activity of essential oils
the repellent activity of essential oils of different plants was reflected in the per cent repellence (table S4). the pooled mean (± SE) repellent activity was significantly different across essential oils (F 90, 4 = 49.1; p < 0.0001) and concentrations (F 90, 2 = 386.6; p < 0.0001). the overall per cent repellence activity was superior in C. deodara (64.76 ± 1.31) over other oils followed by A. calamus (55.05 ± 1.31) and Tagetes minuta (48.76 ± 1.31). the pooled mean repellent activity was significantly higher at 10 mg mL −1 (70.74 ± 1.01%) as compared to other lower concentrations. the mean per cent repellence was also found to be significantly different between the interactions of essential oils and concentrations (F 90, 8 = 13.15; p < 0.0001). the per cent repellence was significantly higher in C. deodara at 10 mg mL −1 (86.29 ± 3.9) as compared to other combinations of treatments and concentrations. the essential oils of Ruta graveolens, Allium sativum, Pogostemom cablin and Tanacetum vulgare showed good repellent activity against Lepidopteron larvae of Cydia pomonella (Landolt et al. 1999). Many researchers also reported the repellent activity of essential oils to Diptera, Coleoptera and thysanopteran pests.

Feeding deterrent activity of essential oils
the feeding deterrence of essential oils against P. xylostella was reflected in the per cent feeding deterrence index (FDI) after 48 h (table S5). the pooled mean (± SE) deterrence activity was found to be significantly different across plants of essential oils (F 135, 4 = 2.91; p < 0.05) and across concentrations (F 135, 2 = 10.30; p < 0.0001). the overall FDI was found significantly superior in A. calamus (42.20 ± 7.05) over other essential oils followed by C. deodara (35.41 ± 7.05) and T. minuta (33.79 ± 7.05). the pooled mean FDI was significantly higher at higher concentration of 10 mg mL −1 (47.48 ± 5.46) as compared to 5 and 2.5 mg mL −1 . However, mean per cent FDI was not differed significantly between the interactions of essential oils and concentrations (F 135, 8 = 0.64; p > 0.05). the present results supported by Jiang et al. (2012) who reported the essential oils of Cymbopogon citratus, C. nardus and C. cassia showed strong feeding deterrent against larvae of Trichoplusia ni. Similarly, asarones isolated from the essential oil of A. calamus rhizomes also showed antifeedant to Peridroma saucia (Koul et al. 1990).

Growth (feeding) inhibition activity
the growth inhibition activity of essential oils was reflected in per cent inhibition after 48 h of feeding (table S6). the pooled mean inhibition activity was found to be significantly different across essential oils (F 135, 4 = 18.76; p < 0.0001) and concentrations (F 135, 2 = 9.34; p < 0.0001). the overall per cent growth inhibition was significantly more in A. calamus (68.55 ± 4.93) followed by C. deodara (52.47 ± 4.93) as compared to others oils. an increase in the concentration of oils resulted in a stronger growth disruption. the overall per cent growth inhibition was significantly higher at 10 mg mL −1 (48.74 ± 3.82) compared to 5 and 2.5 mg mL −1 (40.42 ± 3.82 and 25.69 ± 3.82, respectively). the present results supported by Koul et al. 1990, who reported asarones from A. calamus rhizomes showed potent growth inhibitors to P. saucia.

Reduction in weight gain of larvae due to essential oils
the antifeedant activity of essential oils was reflected in reduction in the larval weight gain after 48 h of feeding (table S7). the pooled mean reduction in weight gain was significantly different across essential oils (F 135, 4 = 15.76; p < 0.0001) and concentrations (F 135, 2 = 23.34; p < 0.0001). the overall (pooled mean) per cent reduction in weight was significantly higher in C. deodara (93.09 ± 6.3) and was followed by A. calamus (80.09 ± 6.3) as compared to other essential oils. the overall reduction in weight was significantly higher at 10 mg mL (83.60 ± 4.88) compared to 5 and 2.5 mg mL −1 (62.26 and 36.51%, respectively). the literature also indicates that insecticidal activities of essential oils depend on degree of absorption, ingestion, inhalation and detoxification capacity of pests in addition to chemical constituents present. the chemical composition of essential oils may differ in relation to plant species, location, season and harvesting period.

Conclusion
Essential oils of C. deodara, A. calamus, M. koenigii and A. marmelos showed residual toxicity to larvae of P. xylostella; whereas, C. deodara and A. calamus showed high repellent activity. A. calamus and C. deodara showed antifeedant activities. C. deodara and A. calamus showed residual toxicity, antifeedant, feeding deterrence, repellence and growth inhibition activities. the insecticidal activities of these essential oils may be due to presence of β-asarone, β-himachalene, Z-ocimene, limonene and α-pinene are major constituents present in the analyzed essential oils.

Supplementary data and research materials
Experimental details relating to this article are available online, alongside tables S1-S7.