INSECTICIDAL ACTIVITY OF QUISQUALIS INDICA L. FLOWER EXTRACT ON SPODOPTERA LITURA FABRICIUS

Anusree S. S.1, Nisha M. S.2 and Sheela M. S.3 1Research Scholar, 2Assistant Professor, 3Professor 1Department of Agricultural Entomology, College of Agriculture, Vellayani 695522. Thiruvananthapuram. Kerala. 2,3Department of Nematology, College of Agriculture, Vellayani 695522. Thiruvananthapuram. Kerala. India Abstract The methanol and ethyl acetate extracts of Quisqualis indica L. flower obtained by crude and soxhlet extraction methods were evaluated for their antifeedant and insecticidal action against third instar larvae of Spodoptera litura Fabricius under laboratory condition. Among the two methods of solvent extraction assessed, crude extractionwas found effective over soxhlet extraction.The results revealed that antifeedant activity was significantly superior in crude methanol extract of Q. indica flower 5 % (31.87 %) compared to other treatments. Maximum insecticidal action (93.51 per cent larval mortality) was recorded in crude methanol extractat 5 % concentrationand it was found as effective as the chemical, quinalphos 0.05 %. Thusthis can beexploited for botanical pesticide production. Key words: Quisqualis indica L., Spodoptera litura Fabricius, Antifeedant bioassay, Insecticidal bioassay, Contact toxicity

I. INTRODUCTION

The world has faced the ill effects of indiscriminate pesticide consumption in agricultural crop productionin the past few decades.Extensive application of chemical pesticides results in serious consequences such as intoxication of people, contamination of water, air and soil, residues in food, resistance in pests and adverse effects on beneficial insects [1]. This demands a search for safer alternatives, which has thrown light into the concept of botanical pesticides.In many parts of the world, locally available were used to manage insect pests as an ancient technology [2]. Plant derived pesticides are eco-friendly, non-toxic to non-target organisms, non-persistent in nature, besides they do not promote resistance. The rich biodiversity of Kerala opens up a wide opportunity to exploit its diverse flora as a suitable alternative to chemical pesticides. Quisqualis indica L. (= indicum (L.) De Filipps), commonly known as Rangoon creeper or Chinese honey suckle is widely used as herbal medicine. It possessed various pharmacological activities such as anti inflammatory, antipyretic, anti oxidant, anti microbial, antihelminthicand immuno-modulatory activities [3]. Far less research has been conducted on its bioactivity against insect pests. The tobacco caterpillar, Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is aneconomically important pestoftropics and subtropics and it is known to causeheavy damage of 25.8 to100 per cent to many of the agricultural crops [4]. The indiscriminate use of chemical pesticides has resulted in field resistance of S. litura to organophosphates, carbamates, pyrethroids etc [5]. This warrants screening of newer molecules of plant origin with unique mode of action which could be a solution for insecticide resistance management. In this context, the present investigation aims to evaluate the insecticidal effects of Quisqualis indica L. against S. litura.

@IJAPSA-2016, All rights Reserved Page 98 International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 10, [October- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X II.MATERIALS AND METHODS

2.1. Extraction of plant material The fresh flowers of Quisqualis indicaL. at full bloom were collected during morning hours from in and around the Instructional farm at College of Agriculture, Vellayani. The plant material was identified from Department of Botany, Kerala University, Karyavattom and the voucher specimen (KUBH – 6010) was deposited at the herbarium of the Institute. The fresh flowers collected were shade dried under room temperature (28±2˚C). The dried flowers were ground thoroughlyinto fine powder. Two methods of extraction viz., crude and soxhlet methods were followed to yield bioactive components from plants. The powdered samples (18 g) were kept in 250 ml ofethyl acetate and methanol in reagent bottlesseparately and were stirredwith a magnetic shaker at room temperature for 72 hours to yield crude extracts of Q. indica.Soxhlet extracts were obtained by extracting the powder (100 g) with respective solvents individually in a soxhlet apparatus.Solutions obtained were filtered and evaporated to air dryness at room temperature and stored in refrigerator at 4˚C till usage. The extract was further made into different concentrations viz. 1.25%, 2.5% and 5% using distilled water for subsequent bioassay. Tween 20 (Polysorbate) at 0.05% was used as emulsifier.

2.2. Collection and rearing of Spodoptera litura The tobacco caterpillar,S. litura collected from Instructional farm, College of Agriculture, Vellayani formed the initial source for continuous, disease free culture. The insect culture was maintained on fresh castor leaves under standard conditions of temperature (28±2˚C) and relative humidity (70±5˚C) throughout the period of study. The laboratory reared larvae were used for different experiments.

2.3. Antifeedant bioassay Antifeedant activity of plant extracts was tested by no-choice method [6]. Fresh castor leaf discs of 5 cm diameter were punched using cork borer and dipped in crude extracts individuallyfor one minute and air dried.Leaf discs treated with water and solvents alone were served as control. Each leaf disc was placed on a filter paper kept over wet padding of cotton in a petri dish (1.5 cm x9 cm) to avoid early dryness of leaf discs.One 3rd instar larvae prestarved for four hours was exposed to each leaf disc.Three such replications were maintained for each treatment.Progressive consumption of leaf area by the larva after 24 hrs feeding was recorded in control and treated discs using Leaf Area Meter and percentage antifeedant activity was calculated according to the formula given by Baskar et al. [7].

2.4. Insecticidal bioassay The crude flower extracts (1 ml) were sprayed separately with different dosages on third instar larvae(20 no’s) using TLC sprayer. Larvae sprayed with 1 ml of the distilled water and solvents alone served as control. Quinalphos 25 EC (0.05%) was used as check. Each treatment was replicated thrice. The sprayed larvae were transferred to rearing bottle and fed with sufficient quantity of fresh castor leaves. Observations were recorded at 24 h interval for 3 days after treatment. Corrected percentage mortality was calculated according to the formula given byAbbott [8].

2.5. Standardization of method of extraction Effective method of solvent extraction was determined from the results of antifeedant and insecticidal bioassay.

2.6. Contact toxicity Dry film method was followed to confirm the contact action ofcrude extracts of Q. indica flower. One millilitre of the extract was poured in each rimless glass test tube and rotated gently. The angles of the test tube was adjusted in such a way that the extract covered three fourth of the inner surface of the test tube. The process was continued till the extract dried up leaving behind a uniform film of extract on the inner surface of glass tube. Third instar larvae of S. litura (20 no’s)

@IJAPSA-2016, All rights Reserved Page 99 International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 10, [October- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X were kept in the glass tube and the mouth of the glass tube was plugged with cotton. The larvae were allowed to remain in contact with the dry film for 4 h. Both distilled water and solvents alone served as control. Each treatment was replicated thrice. Larval mortality was recorded after 4 h. Moribund larvae were also counted as dead one. Percentage mortality was calculated using Abbott’s formula.

2.7. Statistical analysis The experiments were conducted in completely randomized design with three replications. Data after angular transformation was subjected to ANOVA.

III. RESULTS

3.1. Antifeedant bioassay The percentage antifeedant activity ofcrude and soxhlet extracts ofQ. indica flower at 1.25, 2.5 and 5 % concentrations against third instar larvae of S. litura are shown in Table 1

Table-1. Antifeedant activity of solvent extracts of Q. indica flower against S. litura Treatments Mean percentage antifeedant activity

Crude extraction Soxhlet extraction Ethyl acetate 1.25% 13.23±1.23 10.02±0.81

Ethyl acetate 2.5% 20.62±1.92 17.68±1.43

Ethyl acetate 5% 27.24±2.54 22.22±1.80

Methanol 1.25% 16.95±1.58 12.36±1.00

Methanol 2.5% 24.24±2.26 20.35±1.64

Methanol 5% 31.87±2.97 28.80±2.33

Mean 22.36 18.57

Mean of three replications; Mean ± SD Crude methanol extract of Q. indicaflower at 5 % concentration was effective to impart maximum feeding inhibition (31.87 %)to S. litura larvae. Soxhlet methanol extract at 5 % concentration showedantifeedant activity to the extent of 28.80 per cent. The ethyl acetate extracts of Q. indica at higher concentration recorded27.24 and 22.22 per cent antifeedant activity in crude and soxhlet extraction methods respectively.Minimum feeding inhibition (10.02 %) was exhibited by soxhlet extracted ethyl acetate extract of Q. indica flower at 1.25 % concentration. In all the treatments, the antifeedant activity was directly proportional to concentration of the extract. Crude extraction method was found superior to soxhlet extraction method giving feeding inhibition of 22.36 and 18.57 per cent respectively.

3.2. Insecticidal bioassay The solvent extracts of Q. indicaflower were evaluated for their insecticidal actionagainst third instar larvae of S. lituraand the results are presented in Table 2. Table-2. Insecticidal activity of solvent extracts of Q. indica flower against S. litura Mean percentage mortality at different intervals Crude extraction Soxhlet extraction Treatments Hours after treatment Hours after treatment 24 48 72 24 48 72

@IJAPSA-2016, All rights Reserved Page 100 International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 10, [October- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X Ethyl acetate 25.00±1.76 33.26±5.77 39.96±5.00 21.67±2.89 29.92±5.00 34.95±5.00 1.25% (35.22) (39.21) (33.16) (36.24)

Ethyl acetate 41.67±1.70 55.02±5.00 61.69±2.89 28.33±2.89 39.96±5.00 48.31±7.64 2.5% (47.88) (51.76) (39.21) (44.03)

Ethyl acetate 58.33±1.94 71.70±2.89 81.72±2.89 53.33±7.64 60.04±5.00 70.08±5.00 5% (57.86) (64.69) (50.79) (56.84)

Methanol 41.67±3.60 48.31±7.64 56.73±7.64 46.67±7.64 56.73±7.64 63.45±7.64 1.25% (44.03) (48.87) (48.87) (52.80) Methanol 60.00±1.29 70.34±10.00 76.98±7.64 61.67±5.77 68.49±7.64 75.11±5.00 2.5% (57.00) (61.33) (55.85) (60.07)

Methanol 70.00±1.48 86.99±5.00 93.51±2.89 61.67±7.64 76.98±7.64 89.07±7.64 5% (68.86) (75.24) (61.33) (70.69)

Mean 49.45 60.94 68.43 45.56 55.35 63.50

Mean of three replications; Mean ± SD Among the solvent extractsof Q. indica flower evaluated, the most effective treatment observed was crude methanol extract at 5 % concentration, which recorded 93.51 per cent larval mortality after three days of exposure, whilesoxhlet extracted methanolic extract 5 % showed larval mortality to the extent of 89.07 per cent at 72 hours after treatment. Ethyl acetate extract of Q. indicaflower5 % also exhibited 82 and 70 per cent mortality at 72 hours of exposure in crude and soxhlet extraction methods respectively. The larval mortality was found increased with increase of concentration and exposure time.Crude extraction method was found significantly superior to soxhlet extraction method giving larval mortality of 68.43 and 63.50 per cent respectively after three days of exposure.

3.3. Comparison of crude extracts of Q. indica flower with chemical Figure 1. Comparative insecticidal effect of different treatments on S.litura a b 100 a a 90 c c a 80 b b a 70 d b 60 c b d 50 c 40 e c c 30 d 20 d 24 HAT

Percentage mortality mortality Percentage 10 48 HAT 0 72 HAT

Treatments HAT – Hours after treatment

@IJAPSA-2016, All rights Reserved Page 101 International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 10, [October- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X The crude methanolic extract of Q. indica flower 5 % showed70, 86.99 and 93.51per cent larval mortality at 24, 48 and 72 hours after treatment respectively. Quinalphos 0.05 %recordedsignificant larval mortality of 76.67, 90.40 and 99.38 per cent at 24 hours interval for three days respectively. The insecticidal effect exhibited by crude methanolic extract of Q. indica5 % was found statistically on par with quinalphos 0.05 % on first and second day after treatment. 3.4. Contact toxicity Contact toxicity of Q. indica flower crude extractsagainst third instar larvae of S. litura was assessed by dry film method and the results are shown in Figure 2. Figure-2. Contact toxicity of crude extracts of Q. indica flower against S. litura

Ethyl acetate Methanol 55.02 60

50 38.31 34.95 40 26.63 26.63 30 20 9.6

10 Percentage mortality mortality Percentage 0 1.25% 2.50% 5% Concentrations

The bioefficacy study by dry film method recorded maximum larval mortality of 55.02 per cent in crude methanol extract of Q. indica flower at higher concentration of 5 %. Ethyl acetate extract at 5 % concentration showed 34.95 per centlarval mortality against S. litura.

IV. DISCUSSION

Plants are important natural sources of bioactive compounds and many such plant compounds have been included in commercial botanical pesticides.Usage of plant products as pesticide keeps the agricultural produce safe and protects the environment from getting polluted. In the present investigation locally available weed plant, Rangoon creeper, which is known for its pharmacological activities, was evaluated for its bioefficacy against the economically important pest,S. litura. Maximum antifeedant activity was exhibited by crude methanolic extracts of Q. indica flower at higher concentration of 5 % against third instar larvae of S. litura, which could be attributed to the polar nature of phytochemicals responsible for the feeding inhibition. This finding is in agreement with Yogesh et al. [9], whoreportedthe antifeedant activity of crude methanolic extracts ofParthenium hysterophorus L. (69.92 per cent) and Ageratina adenophora (Spreng.) King & H. Rob (63.47%) at 10 per cent concentration against fifth instar larvae of Spodoptera frugiperda J.E. Smith. The crude methanol extracts derived from Q. indica flower at higher dosage of 5 per cent was found highly lethal to third instar larvae of S. litura. Earlier reports are available on the toxicity of Q. indica flower against insect pests.The methanolic extracts obtained from fruits of Q. indica exhibited significant insecticidal activity against four Coccoidea species (Eriococcus lagerstroemiae Kuwana, Ceroplastes japonicas Green., Crisicoccus pini Kuwana and Planococcus citri Risso) [10]. The larvicidal activity of Q. indica flower extracts against S. litura is reported for the first time in this study. The comparison of larvicidal action of plant extracts with synthetic pesticideshowed that the insecticidal action exhibited by methanol extract of Q. indica flower at 5 % concentration till 48 hours after treatment was on par with quinalphos 0.05 %.This fact is supported by Song et al.[11], who recorded 95.70 per cent nymphal mortality in the black pine bast scale,Matsucoccus

@IJAPSA-2016, All rights Reserved Page 102 International Journal of Applied and Pure Science and Agriculture (IJAPSA) Volume 02, Issue 10, [October- 2016] e-ISSN: 2394-5532, p-ISSN: 2394-823X thunbergianae Miller and Park treated with Q. indica extracts and observed that the plant extract was equally effective to the insecticide, fenitrothion 50% EC against M. thunbergianae in field trials. The results obtained from the antifeedant and insecticidal bioassay revealed the effectiveness of crude extraction method compared to soxhlet extraction.This difference in efficacy in extraction methods could be attributed to the heat involved in the extraction procedure. The heating of plant extracts in soxhlet method may cause loss of volatile compounds responsible for bioactivity. A study conducted by Bai[12] reported that the cold extracts of Thevetia nerifolia Juss. exhibited significantly higher mean leaf protection compared to its soxhlet extracts against Henosepilachana vigintioctopunctata Fabricius. The bioefficacy study of crude extracts of Q. indica flower by dry film method confirmed itscontact toxicity against third instar larvae of S. litura.This might be due to the specific mode of action exhibited by the flower extract. Wetwitayaklung et al. [13] reported the acetylcholine esterase inhibition activity of methanolic extracts derived from Q. indica flower in electric eel. But the reports on its mode of action in insects is lacking. Lin et al. [14] reported that flower extracts of Q. indica contained rutin and pelargonidin-3- glucoside, while its seed oil consisted of linoleic, oleic, palmitic, stearic and arachidic acids and a neuroexcitory aminoacid .The bioefficacy exhibited by Q. indica flower extracts could be attributed to its phytochemical constitution. The findings of the present investigation clearly shows that the crude extracts ofQ. indica flower are rich in potential molecules responsible for various bioactivities. The crude methanol extract of Q. indica flower at 5 % concentration was found equally effective as quinalphos 0.05 % against S. litura proving the potential of Q. indica as an ideal substitute for chemical pesticides. Isolation and identification of potential biocidal molecules from this plant could lead to the development of effective botanical pesticides.

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