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Bio-efficacy of Xanthium strumarium L. Essential Oil against Castor Semilooper, Achaea janata L. (Lepidoptera: Noctuidae)

Anuradha K. Shindez1, Rohit S. Parchande1, S.R. Yankanchi1,*
1Department of Zoology, Shivaji University, Kolhapur-416 004, Maharashtra, India.

Background: Although use of synthetic insecticides causes excellent control of insect pests, it leads to adverse effects on non-target organisms as well as the environment. The present study determining the chemical composition and insecticidal activities of essential oil extracted from the leaves of rough cocklebur, Xanthium strumarium and its bioefficacy against the castor semilooper, Achaea janata shall show a new dimension in formulating an ecofriendly management strategy towards castor crop.

Methods: Xanthium strumarium leaf essential oil was isolated through hydro-distillation method using Clevenger apparatus and the chemical composition was analysed using GC-MS. The ovicidal and insecticidal activity of the essential oil was determined by dipping and topical application methods, respectively. Larval feeding deterrence and growth inhibitory properties of the essential oil was evaluated through the diet incorporation method. 

Result: GC-MS analysis of the X. strumarium essential oil revealed the presence of 14 chemical compounds, of which the majority are the terpenes (94.48%). The LC50 values in terms of ovicidal and insecticidal activities were recorded to be 69.70 µL/mL and 0.30 µL/larvae, respectively. The fifty per cent larval feeding deterrence (DC50) and growth inhibitory properties (EC50) were recorded to be 1.88 µl/cm² and 213.94 µL/mL, respectively.

Although synthetic insecticides are the main source for insect pest control, these chemicals have adverse effects on non-target organisms and environment so that leads to search for safe insect control agents. Botanicals could be the possible alternative of synthetic pesticides, which are ecologically sound, economically viable, socially acceptable and environmentally sustainable (Yankanchi and Patil, 2009; Rattan, 2010). Essential oils are the complex mixture of chemicals, lipophilic in nature, easily blended in organic solvents and isolated from leaves, flowers, roots, stems and seeds of plants (Sarma et al., 2019). Out of 17500 plant species belong to the family Asteraceae, Lamiaceae, Myrtaceae, Meliaceae, Lauraceae and Apiaceae possessing essential oils only a limited number (around 300 species) of plants have been utilized commercially in cosmetic and agricultural industries (Pavela and Benelli, 2016; Sharma and Gaur, 2019; Verma et al., 2020; Kouache et al., 2023).

The rough cocklebur, Xanthium strumarium L. (syn. Xanthium indicum) (Family: Asteraceae) is a serious weed common in India, Australia, South Africa and America. It is well known for its medicinal importance in Europe, China, Malaysia and the United States to treat diseases like nasal sinusitis, headache, gastric ulcer, urticaria, rheumatism bacterial, fungal infections and arthritis (Kamboj and Saluja, 2010; Khan et al., 2020). Although, several studies determine the chemical constituents of X. strumarium revealed presence of monoterpenes and sesquiterpenes (Esmaeili et al., 2006; Parveen et al., 2017), but information on insecticidal activities of X. strumarium leaf-based essential oil against lepidopteran insect pests is lacking. Castor semilooper, A. janata is a regular and serious pest on castor during monsoon season in south India and in winter season it spreads in Saurashtra region of Gujrat. Therefore, the present study was designed to determine the chemical composition and insecticidal activities of X. strumarium leaf essential oil against A. janata attacking castor.
Insect culture
The larvae of A. janata were collected from infested castor plants in and around Kolhapur (16°70’N and 74°21’E). The collected larvae were reared on castor leaves at laboratory conditions of 27±2°C, temperature, 75±5% relative humidity (Yankanchi, 2009) in the Department of Zoology, Shivaji University, Kolhapur. Soil was provided for pupation and then collected pupae were kept into insect rearing cage for moth emergence. On emergence, adults were fed with 10% honey solution provided with cotton swab and fresh castor twig with 3-4 leaves was kept in a cage for oviposition. Freshly laid eggs and early third instar larvae so reared were used for the experiments.
Essential oil extraction
Fresh leaves of X. strumarium was collected during July-August, 2022 from Kalamba area of Kolhapur district (16° 63’N and 74°21’E). The plant was identified with the help of voucher specimen MMS-366 deposited in the Department of Botany, Shivaji University, Kolhapur. Essential oil was extracted from one kg fresh leaves through hydro-distillation method using Clevenger apparatus with n-hexane (Wang and Weller, 2006). Extracted oil was concentrated by adding anhydrous sodium sulphate. Oil was collected in airtight glass vials and stored at 4°C for further study. The yield of essential oil was calculated:
Chemical analysis of essential oil
The chemical compounds of essential oil were analysed using GC-MS (Jeol AccuTOF GCV) fitted with a flame ionization detector and capillary column (30 M × 0.32 MM, 0.25 µm) at the Sophisticated Analytical Instrument Facility (SAIF), IIT, Powai, Mumbai. Helium was used as a carrier gas with flow rate of 0.1mL/min and a splitless injection program of 60-1M-6-200-1M-8-270-10M-5-280-0. The temperature of the oven was kept for 1 min at 60°C and raised to 200°C at a rate of 6°C/min and then raised to 270°C at a rate of 8°C/min. Later, the temperature was held constant at 270°C for 10 minutes and then increased to 280°C at a rate of 5°C/minute with a total run time of 30 minutes. The ionization voltage for the electron impact (EI) mass spectra was 70 Ev and the scan range of mass spectra was 350-800 m/z. The identification of compounds was performed by comparison of their retention time with the spectrum of known compounds in NIST MS 2.0 minlib to find out the names, structure, molecular weight and molecular formula. The relative percentage of each chemical component was derived by peak area normalization.
Ovicidal activity
All experiments were conducted in the Department of Zoology, Shivaji University, Kolhapur during 2022-23. Ovicidal activity of essential oil was determined using the dipping method proposed by Devarshi et al., (2017). Essential oil was serially diluted in acetone to get the desired concentration viz., 10, 20, 40, 80 and 160 µl/mL of acetone. Freshly laid 20 eggs were glued on a paper strip and dipped in each concentration of essential oil for a minute and control eggs were dipped in acetone. For each concentration, there were three replicates. After evaporating the solvent, both treated and the control eggs were kept in solo cups lined with wet filter paper under laboratory conditions. The eggs were observed daily until all eggs were hatched in control. Per cent ovicidal activity (OA) was calculated using following formula:
C and T = Number of eggs hatched in control and treatment, respectively.
Insecticidal activity
Insecticidal activity of X. strumarium essential oil was evaluated by a topical application method as described by Pavela (2005). Freshly molted third instar (11-12 mg) larvae placed in a petri plate lined with filter paper. A total of 2 µL acetone mixed with 0.05, 0.1, 0.2, 0.4 and 0.8 µL of essential oil was applied to the dorsal side of each larva with a micropipette and control larvae received the same volume of acetone. Each treatment was replicated thrice with 10 numbers of larvae per replication. The treated and untreated larvae were placed on petri plates (Borosil 90 mm dia.) lined with moist filter paper and a castor leaf for feeding. Larval mortality was recorded at 24 and 48 hrs after treatment, wherein moribund larvae were considered to be dead.  
Feeding deterrence activity
The feeding deterrence activity of essential oil was determined with the no-choice method proposed by Isman et al., (1990). The castor leaf discs (1.5 cm dia., 1.76 cm2 area) were cut using a cork borer. Five concentrations (0.2, 0.4, 0.8, 1.6 and 3.2 µL/ mL) of essential oil were prepared in acetone. Essential oil was applied to both sides of the leaf disc and the control disc applied the acetone. After evaporation of the solvent, a leaf disc was kept in a Petri plate lined with wet filter paper to avoid leaf drying. Early third instar larvae were starved for 2-3 hours prior to experiments. A starved larva was released into the center using a camel brush to feed. There were fifteen replicates to each concentration and control. Experiments were terminated when more than 50% of the control disc were eaten. Treated and control discs were photographed and were measured using Image-J software. A feeding deterrence index (FDI) was calculated using the formula:
C and T = Leaf area consumed by the larva of control and treated discs, respectively (Akhtar et al., 2010) and results were expressed in µL/cm2.

Growth inhibitory activity
Larval growth inhibitory activity of essential oil was determined as per the method described by Reddy et al. (2016). Five concentrations of essential oil were prepared as 20, 40, 80,160 and 320 µL/ mL of acetone and applied on both sides of castor leaf discs. Only acetone was applied on the control leaf disc for comparison. Treated and controlled leaf discs were kept in a Petri plate lined with wet filter paper and then pre-weighted early third instar larva was released to feed. There were fifteen replicates in each concentration including control. After 48 hrs, each concentration and control larva were weighted and then larval growth inhibition (GI) was determined by using the formula:
C and T = Larval weight in control and treatment, respectively. (Guo et al., 2014).
                 Growth inhibitory results were expressed in µL/mL of acetone.  
Statistical analysis
All experimental data were analysed using descriptive statistics and the LC50 value was calculated through probit analysis (Finney, 1971) using SPSS software (version 21.0). The 50% feeding deterrence concentration (DC50) and 50% effective concentration for reducing the larval growth (EC50) values were calculated by substituting the value of x in the regression equation:
Y = B x + A
B = Slope of the line.
A Y= Intercept and Y set to 50% to calculate EC50 and DC50 values).
The essential oil obtained by hydro-distillation from X. strumarium leaves were yellowish in colour with an average yield of 0.23% (v/w). The yield of essential oil was found varied from 0.12 to 0.35% (v/w) in the previous reports (Taher et al., 1985; Parveen et al., 2017), which principally depends upon plant parts, harvesting season, plant polymorphisms, type of soil, fluctuation in precipitation, temperature, day duration and light frequency (Pavela and Benelli, 2016)

The gas chromatography coupled with mass spectrometry analysis of X. strumarium leaves essential oil revealed presence of 14 compounds. The presence of terpenes was more (94.48%), of which 14.33% monoterpene hydrocarbons, 72.01% sesquiterpene hydrocarbons and 8.14% oxygenated diterpenes (Table 1). Essential oil extracted from plant leaves collected from Iran showed presence of 28.8% monoterpene hydrocarbons, 47.2% sesquiterpene hydrocarbons and 17.9% oxygenated monoterpenes (Sharifi-Rad et al., 2015). The essential oil of X. strumarium leaves collected from different geographical localities showed variations in compounds for instance Scherer et al. (2010) observed the 24 compounds and 22 compounds recorded by Esmaeili et al., (2006). The qualitative variations in essential oil composition of plant materials are influenced by the genetic and ecological factors (Sharifi-Rad et al., 2015).   

Table 1: Chemical composition (%) of X. strumarium leaf essential oil.

As the data on ovicidal and insecticidal properties of X. strumarium leaf essential oil against A. janata eggs and early third instar larvae revealed dose-dependent variation as against no toxicity in the control. The LC50 value of ovicidal (69.70 µL/mL) and insecticidal (0.30 µL/larva) properties were presented in Table 2 and the chi-square values were significantly different at P≤0.05. Essential oil was able to inhibit the embryonic growth as well as kill the early instar larvae of A. janata and present results are confirmed with a previous report of Omar (2012). He has observed that X. strumarium fruit ethanol extract significantly inhibits the egg hatchability and adult mortality of khapra beetle, Trogoderma granarium. The fruits and leaves extract of X. strumarium showed the insect repellent activity against Colorado potato beetle, Leptinotrasa decemlineata (Cetinsoy et al., 1998), insecticidal effects on green peach aphid (Erdogan and Yildirim, 2016) and stored grain insect pests (Roy et al., 2014; Yohannes et al., 2014).

Table 2: Toxic activities of X. strumarium essential oil against early third instar larvae of A. janata.

Feeding deterrent and growth inhibitory activities of X. strumarium essential oil results are given in Fig 1 and 2. The concentration of essential oil showed positive relation for feeding deterrence (R2 = 0.8794) and growth inhibitory (R2 = 0.8805) activity. Essential oil was affected to A. janata larvae by both assays in dose response manner. Fifty percent deterrence concentration (DC50) was 1.88 µl/cm² and effective concentration for fifty per cent (EC50) was 213.94 µL/mL to A. janata. Solvent extract of X. strumarium leaves recorded highest antifeedant activity against mosquito bug, Helopeltis theivora (Sarmah and Bhola, 2014) and leafroller, Choristoneura rosaceana (Gokce et al., 2011). Sarmah et al., (2007) observed the substantial results of ovicidal, post-embryonic development and adulticidal activities of X. strumarium leaf solvent extracts against red spider mite, Oligonychus coffeae as compared to the control. Generally, the essential oil contains more terpenes that are lipophilic in nature so they interfere with biochemical processes, causing physiological imbalance in insects. A number of reports proved that the toxic action of essential oils is due to inhibition of acetylcholinesterase activities (Loh et al., 2021; Hung et al., 2022). Insect toxicity is related to essential oil lipophilicity and the higher the lipophilicity, the better penetration of oil molecules into the cuticle and these factors might have contributed to oil toxic effects in the insects (Pavela 2012; Parchande et al., 2023).

Fig 1: Effect of X. strumarium essential oil on feeding deterrence against A. janata.


Fig 2: Effect of X. strumarium essential oil on larval growth inhibition (%) of A. janata.

The essential oil of X. strumarium leaves showed ovicidal, insecticidal, antifeedant and growth inhibitory properties against castor semilooper, A. janata, which could be utilized for the development of bioinsecticides for the management of A. janata, substituting the conventional chemical insecticides.
Anuradha Shinde is grateful to the Chhatrapati Shahu Maharaj Research, Training and Human Development Institute (SARTHI), for fellowship (Ref. No. SARTHI/Fellowship/CSMNRF-2021/2021-22/896). The authors are thankful to Shivaji University, Kolhapur for the financial assistance (Ref. No. SU/C.andU. D. Section/299 dt. 01/11/2021). The authors wish to thank Common Facility Centre (CFC), IIT Bombay for the GC-MS analysis.
We declare that we have no conflict of interests.

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