Phytochemical and insecticidal activity of Euphorbia pulcherrima willed extracts for controlling cotton leaf worm, Spodoptera littoralis Boisd

Heba Youssif El-Sayed Ibrahim^1*, Abdel-Raouf Mohamed Sallam², Fatma Jamal Salh Hamdi³

¹ Plant Protection Research Institute, Agriculture Research Center (ARC), Dokki, Giza, Egypt

² Professor of Entomology, Zoology Department, Faculty of Science, Mansoura University, Mansoura, Egypt

³ Faculty of Science, Zoology Department, Tripoli University, Tripoli, Libya

Abstract

Chemical constituents of Euphorbia pulcherrima Willed. Aerial parts were extracted and tested for their insecticidal activity against different stages (eggs, 4^th instar larvae and pupae) of Spodoptera littoralis Boisd. Under laboratory conditions. The most effective fraction against all stages was petroleum ether fraction with LC₅₀s of: 591.14, 859.90 and 3004.44 ppm for eggs, 4^th instar larvae and pupae, respectively. Also, the volatile constituents of each extract fraction were qualitatively and quantitatively characterized and identified by Gas Chromatography-Mass Spectrometry technique. E. pulcherrima extracts showed promising activity as green alternatives of traditional insecticides.

Keywords: Euphorbia pulcherrima, extracts, phytochemical, insecticidal activity and Spodoptera littoralis

Introduction

The cotton leaf worm, Spodoptera littoralis (Boisd.) (Lepidoptera; Noctidae) is one of the most destructive polyphagous agricultural pests. It attacks many economically crucial crops such as cotton, tomato, maize and vegetables as well as many kinds of fruits ^{[1, 2, 3]}. It harm the plants directly by extensive feeding of the larvae on plant leaves or bear into the buds for consumption ^[4]. Also, the presence of the larvae or their faeces reduces the marketability of vegetables and ornamentals ^[5]. The extensive and intensive use of broad-spectrum synthetic insecticides against this serious pest led to the development of resistance to many registered insecticides ^[6]. Moreover, problems such as residuals toxicity and harmful effects on beneficial insects were raised. So, the global public concern of looking for environmental friendly alternatives for insect control was increased. Phytoinsecticides are considered promising agents in this trend.

 Many plants belonging to various families were studied for searching for novel insecticidal components ^{[7, 8, 9]}. Euphorbiaceae family, is well known for its diverse characteristic content of phenolic compounds (phenolic acids, tannins, coumarins, flavonoids, lignans, phenanthrenes, quinones, etc…), triterpenoids and associated compounds (alcohols, sterols and hydrocarbons), alkaloids, cyanogenic glucosides and Glucosinolates ^[10]. E. pulcherrima contains numerous chemical groups such as terpenoids, alkaloids, flavonoids, steroids, saponins, glycosides, reducing sugars ^{[11, 12]}.

The objective of this study was to investigate the phytochemistry and the insecticidal activity of E. pulcherrima aerial parts extracts against different stages of the cotton leaf worm, S.littoralis.

Materials and Methods

Plant Material

E. pullcherima aerial parts were collected in November 2019 from garden of Mansoura University, Egypt. Plant identification was confirmed by department of Botany, Faculty of science, Mansoura Univ., Egypt.

Extraction and phytochemical screening of E. pulcherima

Aerial parts of the plant were dried at room temperature and grounded to a powder, then, soaked in methanol three times, each time for three days. Methanolic extract of E. pulcherrima was filtered, evaporated to approximately 1/3 of its volume, then diluted with distilled water and successively extracted by different organic solvents of ascending polarity (petroleum ether, methylene chloride and ethyl acetate) by using separating funnel. Anhydrous sodium sulphate was used for drying all fractions, then, they were evaporated to dryness. Each fraction was formulated in serial concentrations to evaluate its insecticidal activity against different stages of S. littoralis.

Phytochemical screening

Volatile components of E. pulcherrima extracts were characterized by GC/MS technique using a TRACE GC Ultra Gas Chromatographs (THERMO Scientific Corp., USA), coupled with a thermos-mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC-MS system was equipped with a TR-5 MS column (30 m x 0.32 mm i. d., 0.25 μm film thickness). Helium was used as a carrier gas at a flow rate of 1.0 mL/min and a split ratio of 1:10 using the following temperature program: 60 C for 1min; rising at 4.0 C/min to 240 C and held for 1min. The injector and detector were held at 210^oC. Diluted samples (1:10 hexane, v/v) of 1μL of the mixtures were always injected. Mass spectra were obtained by electron ionization (EI) at 70 eV, using a spectral range of m/z 40-450. Identification of the chemical components were de-convoluted using AMDIS software (www.amdis.net) and identified by its retention indices, mass spectrum matching to Wiley spectral library collection and NSIT library database.

The tested insect pest

Susceptible strain of S. littoralis was obtained from Plant protection Research Institute, Agricultural Research Center, Giza, Egypt. It was reared under laboratory controlled condition (27± 2^oC, 65±5% R.H). Larvae were maintained on fresh castor leaves till pupation. Pupa were collected and kept in clean jars till emergence of adults. The emerged adults were fed on 10% honey solution on cotton wick.

Bioassay

Assessment of ovicidal activity of different fractions of E. pulcherrima extract

Newly egg masses of S. littoralis were sprayed by the formulated emulsions of E. pulcherrima fractions containing 0.3% Tween-80. Each treatment was replicated three times. Another three replicates were sprayed only with water and 0.3% Tween80 to act as control.

Assessment of insecticidal activity of different fractions of E. pulcherrima against S. littoralis 4^th instar larvae

Fresh castor leaves were cleaned and sprayed with different concentrations of each extract fraction and given to the larvae. Each concentration was represented by three replicates, each of them contain 10 larvae. In addition to the three replicates of control (water + Tween 80).

Assessment of insecticidal activity of different fractions of E. pulcherrima against S. littoralis Pupae

Newly formed pupae of S. littoralis were sprayed with different fractions concentrations. After 10 days, the dead pupae or that failed in emerging adults were counted.

Statistical analysis

Mortality percentages of the tested instar were estimated and corrected by using Abbott`s formula ^[13]. The LC₅₀, LC₉₀ values and slope were determined according to Finney, (1971) ^[14]. Also, toxicity index of each tested fraction was estimated using Suns' equation ^[15].

Results and Discussion

Phytochemical screening

The volatile components of different fractions of E. pulcherrima aerial parts were characterized by GC/MS technique "Table 1". It was found that pet ether fraction revealed 6 compounds represented by 6 peaks, while methylene chloride fraction revealed 22 compounds. Also, the major volatile components of ethyl acetate fraction were seven compounds corresponding to seven peaks.

Table 1: The GC/MS analysis of different fractions of E. pulcherrima aerial parts

Insecticidal activity

Ovicidal activity of E. pulcherrima extracts on S. littoralis eggs

Data in "Table 2" showed that the hatchability of S. littoralis eggs was significantly suppressed by all fractions of E. pulcherrima extracts. Petroleum ether fraction was the most efficient showing the highest ovicidal activity followed by methylene chloride fraction then ethyl acetate fraction with LC₅₀: 591.14, 686.18 and 1678.76 ppm, respectively. The absolute reduction of the eggs hatching was recorded at 10000 ppm of both of petroleum ether and methylene chloride fractions and at 20000 ppm of ethyl acetate fraction. Egg masses failed to hatch without any morphological changes. So, the ovicidal activity of the tested fractions may be occured by disrupting or inhibiting embryogenesis obstructing their hatch ^[16].

Table 2: Ovicidal effects of E. pulcherrima extracts against S. littoralis eggs.

Insecticidal activity of different E. pulcherrima extracts against S. littoralis 4^th instar larvae Data in "Table 3" showed that the most effective fraction against S. littoralis 4^th instar larvae was petroleum ether fraction followed by ethyl acetate then methylene chloride with LC₅₀: 859.91, 1291.97 and 2515.79 ppm, respectively. All tested fractions exhibited shrinkage of the larvae bodies, loss of body weight and abnormal pupation before the normal time. Ethyl acetate extract turned the color of larvae to black and the body takes out its liquid contents. The present data was consistent with Almeida et al., (2017) ^[17] who reported growth inhibition and poor weight of S. frugiperda 2^nd instar larvae treated with the ethanolic extract of E. pulcherrima. It was argued that the loss or poor weight of the larvae attributed to feeding and metabolism reduction ^[18].

Table 3: Insecticidal activity of different fractions of E. pulcherrima extract against S. littoralis 4^th instar larvae

Insecticidal activity of different E. pulcherrima extracts against S. littoralis pupae

Data in "Table 4" showed that S. littoralis pupae were less susceptible to the all tested fractions of E. pulcherrima extract than larvae. Petroleum ether fraction kept the first rank showing the highest efficacy against S. littoralis pupae, followed by ethyl acetate fraction then, methylene chloride fraction with LC₅₀s: 3004.44, 3984.94 and 5904.67 ppm, respectively. Also, there was a great reduction of pupae weights comparing to the control to the extent that it looks as if it were empty especially when treated with petroleum ether fraction. Ethyl acetate extract turned the color of pupae to a dark black with solidified secretions at the pointed end of the pupae. The current data emphasized that all fraction of E. pulcherrima extract have a contact effect and having the ability to penetrate the cuticle of pupae hindering the emergence of adults.

Table 4: Insecticidal activity of different fractions of E. pulcherrima extract against S. littoralis pupae