Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the dominant insect pest of cruciferous crops around the world, and is resistant to many chemical insecticides. In this study, we measured the chemical composition and bioactivity of Artemisia lavandulaefolia DC (Asteraceae) essential oil on P. xylostella. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography-mass spectrometry. A total of 35 constituents were identified. The principal compounds were: eucalyptol (35.60%), (R)-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol (16.25%), π-trimethyl-3-cyclohexene-1-methanol (6.83%), 3-methyl-6-(1-methylethyl)-2-cyclohexen-1-one (6.63%), and (1S)-1,7,7-trimethyl-bicyclo[2.2.1] heptan-2-one (4.72%). The LD50 contact toxicity of the essential oil to immature P. xylostella was estimated at 0.045 μL per larva. Artemisia lavandulaefolia oil exhibited fumigant toxicity against P. xylostella adults with an LC50 of 0.113 mg per L after 12 h and also provided 80 to 100% repellency at a 1% v/v concentration.
Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the most important insect pest of cruciferous crops throughout the world (Javler 1992). The annual cost for managing this pest has been estimated to be US$1 billion (Talekar & Shelton 1993). However, recent data on management costs combined with associated crop losses by diamondback moths have been reported to be US$4–5 billion (Zalucki et al. 2012). Chemical control of P. xylostella has become less effective because of the diversity and abundance of host plants, lack or disruption of natural enemies, high reproductive potential (up to 20 generations per year), and proven ability to rapidly evolve resistance to insecticides used for its control (Magaro & Edelson 1990; Lim et al. 2002; Liang et al. 2003; Furlong et al. 2013; Lu & Lee 1984; Khan et al. 2005). Long-term use of synthetic insecticides has given rise to many ecological problems, including residues that are toxic to wildlife, and possible bioaccumulation issues associated with the environment (Shelton et al. 1993; Charleston & Kfir 2000; Isman 2006). Bioactive plant-derived compounds have been suggested to be alternative sources for insect control because many are selective to insect pests and have no or minimal adverse effects on non-target organisms and the environment (Regnault-Roger 1997; Walter 1999; Schmutterer 1990; Prakash & Rao 1996; Charleston et al. 2005).
Artemisia lavandulaefolia DC (Asteraceae) is a perennial herb with procumbent rhizomes while the aerial components are strongly aromatic. This plant species has been used in traditional medicine in many cultures for treatment of stomatitis, fever, bronchitis, chronic cervicitis, hemorrhagia, cholecystitis, including some cardiovascular diseases and liver ailments (Wang et al. 2006a; Cha et al. 2010).
The essential oil of A. lavandulaefolia contains various chemicals proven to inhibit the mycelial growth of Pyricularia grisea (Pyriculariaceae) and Rhizoctonia solani (Ceratobasidiaceae) fungi (Jiang et al. 2008) as well as possessing antimicrobial activity against obligate anaerobic bacteria (Cha et al. 2005). The chemical composition of A. lavandulaefolia essential oil has been characterized in several studies and whole plant extracts were found to have insecticidal activity on Sitophilus zeamais (Motsch.) (Coleoptera: Curculionidae) (Yuan et al. 2010; Liu et al. 2010a). Also, numerous studies have reported the bioactivity of essential oils derived from various Artemisia species against stored-product insects (Kordali et al. 2006; Liu et al. 2006, 2010b; Wang et al. 2006b; Goel et al. 2007; Negahban et al. 2007; Tripathi et al. 2000; Chu et al. 2010, 2012; You et al. 2015). However, no studies have evaluated the bioactivity of A. lavandulaefolia against P. xylostella. Therefore, we report here on the contact and fumigant toxicity, as well as repellent properties of A. lavandulaefolia essential oil, on P. xylostella in laboratory trials.
Materials and Methods
PLANT MATERIAL AND ESSENTIAL OIL EXTRACTION
Fresh aerial parts of A. lavandulaefolia were collected in Sep 2015 at the flowering stage in Changchun (43.8170°N, 125.3235°E), China. Plant samples were dried in the shade at ambient temperature, then crushed and soaked in water for 12 h with a solid: liquid ratio of 1:1, Afterwards, the crushed aerial parts were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus. The oil was dried over anhydrous sodium sulfate and stored in a sealed vial in a refrigerator at 4 °C.
Plutella xylostella were reared from larvae and pupae obtained from cabbage in an experimental field of Jilin Agricultural University, Changchun, China. Larvae were reared on individual cabbage (Brassica oleracea var. capitata) plants that had never been exposed to pesticides, and maintained in a screened cage (40 × 29 × 17 cm) at 25 ± 1 °C and 75% RH, and a 12:12 h (L:D) photoperiod. After moth emergence, the adults were fed a 10% honey solution. Cabbage leaves were used for oviposition. Third instars and 3-day-old adults were used in bioassays.
GAS CHROMATOGRAPHY AND MASS SPECTROMETRY ANALYSES
The essential oil of A. lavandulaefolia was analyzed using a gas chromatograph (Agilent 6890N, Agilent Technologies Incorporated, California, United States), and the oil constituents were identified using a mass spectrometer (MS, Agilent 5975N, Agilent Technologies Incorporated, California, United States). The gas chromatograph apparatus was equipped with an HP-5 capillary column (30 m × 0.25 μm inside diameter, film thickness of 0.25 μm). Settings were as follows: initial column temperature held at 60 °C for 3 min, then ramped at 10 °C per min intervals to 180 °C and held isothermally for 1 min, and finally raised to 280 °C at 20 °C per min and maintained for 5 min. The injector temperature was maintained at 280 °C. A diluted 1 μL sample of essential oil was injected at a split ratio of 50:1. Helium was used as the carrier gas at a flow rate of 1.0 mL per min. The mass spectrometer spectra used an electron ionization source (70 eV ionization, source temperature of 230 °C). The scan range was 20-650 m/z at 2 scans per s. Constituents of the essential oils were identified by comparing the results with the mass spectra libraries (National Institute of Standards and Technology, Gaithersburg, Maryland, USA: NIST databases), and component relative percentages were expressed as percentages by peak area normalization (Adams 1989).
Third instar P. xylostella were used to evaluate the contact toxicity of the essential oil. Five concentrations (0.025, 0.05, 0.075, 0.1 and 0.125 μL per larva) were diluted in acetone. All treatments used a 0.5-μL dose to the dorsal thoracic region. Acetone was used as a control. Ten larvae were treated per concentration, and the study was repeated 3 times. Treated and control insects were placed separately in Petri dishes (90 mm diam) and kept in incubators at 29 ± 1 °C and 75 ± 5% RH for 24 h, after which mortality was recorded during 2, 4, 6, 8, 10, 12, and 24 h. Mortality was calculated as follows:
Three-day-old adults of P. xylostella were used to evaluate the fumigant toxicity of the essential oil. Serial dilutions of the A. lavandulaefolia essential oil were treated with acetone (0.1, 0.2, 0.3, 0.4, and 0.5 mg per L). Acetone was used as a control. Ten μL of the appropriate concentration of the essential oil was added to filter paper (8.0 cm × 1.5 cm). The solvent was allowed to evaporate for 30 s before the cap was placed on the glass bottle (60 mL, with 10 insects) to form a sealed chamber. All treatments and controls were maintained in incubators (29 ± 1 °C, 75 ± 5% RH). The mortality was recorded during 2, 4, 6, 8, 10, and 12 h.
The repellent activity of the essential oil to individual P. xylostella adults was measured using a “Y” glass tube olfactometer. The essential oil was tested at different volume fractions (0.25, 0.5, 1, 2, and 4%. v/v) in acetone. Each tube was connected to an aromatic-source bottle where 10 μL of the appropriate concentration was added to a 25 × 10 mm filter paper, then placed in an aromatic-source bottle after the solvent evaporated for 30 s. Acetone was used as a control. A fluorescent light was set parallel above the Y-tube to avoid light interference. Both arms of the tube were filled with pure humidified air at a rate of 400 mL per min.
A single adult diamondback moth was placed at the entrance of the olfactometer and after 10 min, its position in the tube was recorded (Wang et al. 2016). Moth response criteria were determined as follows: if the moth climbed to more than half the length into one of the tubes and remained for 1 min or more, it was deemed the insect chose this path; if the moth made no choice after 5 min, it was deemed no choice. Ten adults were exposed to each concentration and each concentration was replicated 3 times. The olfactometer tube was cleaned with ethyl alcohol after each concentration. The percent repellency (PR) values were determined as follows:
Statistical procedures for all analyses used SPSS Statistics 17.0 (IBM, New York, New York, USA). Results from all bioassays were subjected to probit analysis to determine the LC50 and LD50 values, fiducial limits, and slopes (Alarie 1988; Sakuma 1998). To determine if differences (P < 0.05) existed between treatments and controls in repellent bioassays, data were analyzed using Student's t-test. These data were plotted by Prism 6.0 (GraphPad Software, La Jolla, California, USA).
GAS CHROMATOGRAPHY AND MASS SPECTROMETRY ANALYSES
The essential oil yield of A. lavandulaefolia was 4.50 × 10-3 L per kg (v/w). A total of 35 components were identified, accounting for 97.37% of the total oil (Table 1). The main compounds were eucalyptol (35.59%), (R)-4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol (16.25%), π-trimethyl-3-cyclohexene-1-methanol (6.82%), 3-methyl-6-(1-methylethyl)-2-cyclohexen-1-one (6.63%), and (1S)-1,7,7-trimethyl-bicyclo[2.2.1]heptan-2-one (4.71%).
In the contact toxicity study, the LD50 of A. lavandulaefolia essential oil was 0.07 μL per P. xylostella larva at 2 h and 4 h post application then subsequently decreased to 0.05 μL per larva at 24 h (Table 2). For the fumigation toxicity study, the LC50 of the essential oil was greatest at 0.25 mg per L for adult diamondback moths at 2 h exposure (Table 3). The effectiveness of this concentration decreased with time to an LC50 of 0.113 mg per L after 12 h of continuous exposure. Artemisia lavandulaefolia oil produced its greatest repellency to adult P. xylostella at the 4% concentration. However, only the repellency of the 0.25% concentration proved to be significantly different from controls (Fig. 1).
Chemical constituents of Artemisia lavandulaefolia essential oil.
The chemical compositions of the essential oil reported here are in partial agreement with previous reports, as Deng et al. (1987) reported that the main constituents of A. lavandulaefolia essential oil to be eucalyptol (36.54%), borneol (3.50%), and 4-terpineol (2.59%). Yuan et al. (2010) reported that the principal compounds of the essential oil from this plant species when extracted by steam distillation contained eucalyptol (10.74%), α,α,4-trimethyl-3-cycloexene-1-methanol (5.26%), and 4-carene (4.00%). Zhang et al. (2012) reported that eucalyptol (20.62%), borneol (15.32%), and eudesm-7(11)-en-4-ol (13.81%). Indeed, variation in chemical composition of essential oils may be due to geographic and seasonal factors. For example, the main compounds of the essential oil of A. lavandulaefolia collected from Jiangxi Province (Northern China) were caryophyllene (15.53%), (1R)-1,7,7-trimethylbicyclo[ 2.2.1]heptane-2-one (10.37%), α-caryophyllene (8.8%), camphor (6.89%) and D-myrcene (6.48%) (Xiong 2011). In addition, the main compounds of the essential oil obtained from Beijing (Central China) were caryophyllene (15.5%), β-thujone (13.8%), eucalyptol (13.1%), and β-farnesene (12.3%) (Liu et al. 2010a). The compositions in the oil from Guizhou Province (Southern China) were caryophyllene (25.39%), 7,11-dimethyl-3-methylene-1,6,10-dodecatriene (13.21%) and rysanthenone (7.75%) (Ma et al. 2012).
Contact toxicity of Artemisia lavandulaefolia essential oil to Plutella xylostella larvae.
Fumigation toxicity of Artemisia lavandulaefolia essential oil to Plutella xylostella adults.
In summary, our study showed that extracts of the aerial portion of A. lavandulaefolia possessed contact toxicity, fumigant, and repellent activity against P. xylostella. Additionally, we believe that the essential oil from this plant species has potential for development as a novel bioactive product against P. xylostella. Further studies are required to characterize those components of the essential oil with the greatest bioactivity for additional screening, so that their potential application in controlling pests can be fully exploited.
We are thankful to our colleagues for their assistance in plant specimen collection and insect rearing. This work was supported by the National Natural Science Foundation of China (31101440), Quality and Safety of Agricultural Products Program (2011-Z37), and the National University Students Innovation and Entrepreneurship Training Program of Jilin Agricultural University (201410193002 and 201410193004). All the authors are especially grateful to the Insect Department of College of Agronomy, Jilin Agricultural University, for providing laboratory facilities.