Antifeedant, larvicidal and ovicidal activities of fractions isolated from ethyl acetate crude extracts of Barleriabuxifolia leaves were tested against fourth instar larvae of Spodopteralitura and Helicoverpaarmigera. The maximum antifeedant, ovicidal and larvicidal activity was recorded in fraction III of B. buxifolia against S. litura and H. armigera. Whereas significant larval mortality was observed in fraction III of B. buxifoliaon S. litura (78.66%) and H. armigera(73.76%) at the same concentration. These results indicate that B. buxifolia has the potential to serve as an alternate botanical pesticide in the management of Spodopteralitura and Helicoverpaarmigera.
Keywords: Antifeedant, Insecticidal, Ovicidal activities, Spodopteralitura, Helicoverpaarmigera, Barleriabuxifolia.
Received: 14 April 2017 / Revised: 18 May 2017 / Accepted: 23 June 2017 / Published: 20 July 2017
This study to approach the novel aspects of plant phytochemicals act as insecticides against economically important pest. Fractions isolated from B. buxifoliaand tested for insecticidal activity on S. litura and H. armigera is new report in this plant. Further, it may identify the active principles which may use as potential plant derived insecticide.
The environmental problems caused by overuse of pesticides have been the matter of concern for both scientists and the public in recent years. It has been estimated about 2.5 million tons of pesticides are used in crop protection for each year and the worldwide damage caused by pesticides reaches 100 billion annually [1 ]. Due to a higher dose and repeated frequency of application, every year one million people suffer from pesticide poisoning, cardiopulmonary, neurological and skin disorders, fetal deformities, miscarriages, lowering the sperm count of applicators. Insect pests play a major role in damaging the agricultural crops and the loss varies between 10% and 30% for major crops [2 ]. In india, Spodopteralitura Fabricius (Lepidoptera: Noctuidae) is one of economically important insect and it damages many economically important crops including cotton, pigeonpea, chickpea, tomato, okra, and black gram [3 ]. The cotton bollworm, Helicoverpaarmigera (H. armigera) (Hübner) (Lepidoptera: Noctuidae) is a polyphagous pest worldwide that inflicts crop damage in India to the sum of one billion dollars annually and it attacks over 200 crop species belonging to 45 families [4 ].These pests status is well justified in its polyphagy on all economically important crops and the hurdles in its management. These insect pests have been controlled with the help of synthetic insecticides over the past fifty years [5 ].
Botanical pesticides provide an alternative to synthetic pesticides because of their generally low environmental pollution, low toxicity to humans and other advantages. While plant chemicals may produce toxic effects when ingested by insects, antifeeding activity may determine the extent of insect herbivory. Several papers have been published on the entomotoxic properties of crude extracts from different plant species [6 , 7 ]. Plants are endowed with a potential to produce a range of secondary metabolites like alkaloids, terpenoids, flavonoids, these phytochemicals are known to protect the plants from the attack of insect-pests. Phenols, glycosides, sitosterols and tannins.Solanummelongena., Lycopersiconesculentum And Capsicum annuum. (Solanaceae) are widely cultivated in India and other parts of the world. Few reports are available using C. annuumfruit powder [8 ]. However, primary work on Barleriabuxifoliabiological properties against agricultural insect pests has been already reported [9 ]. Further, the present investigation was carried out to evaluate the antifeedant, insecticidal and growth inhibitory activities of isolated fractions of Barleriabuxifolia against economically important pests.
2.1. Collection of Plant Materials
The leaves of Barleriabuxifolia were collected from Pulliansolai, Kolli hills, namakkal District, Tamil Nadu, India during the July 2015. Collecetd plant specimen was identified by Dr. S. John Britto, Director, The Rapinat Herbarium and Centre for Molecular Sytematics, St’ Joseph’s College, Tiruchirapalli, Tamil Nadu, India and The Voucher specimen (IPH 16) was deposited in Entomology lab, Arignar Anna Government Arts College, Musiri, Tamil Nadu, India. The plant leaves were carefully washed with clean water and shade dried under room temperature (27.0 ± 2°C) at Entomology lab, PG & Research Department of Zoology, Arignar Anna Government Arts College, Musiri, Tamil Nadu, India.
2.2. Extraction and Fractionation
The plant materials were thoroughly washed with tap water and shade dried under room temperature (27.0± 20C and 75 ± 5% RH). After complete drying the plant materials were powdered using electric blender and sieved through a kitchen strainer. 1000g of plant powder was extracted by soxhlet extraction methods with ethyl acetate solvent and filtered through Whatman’s No. 1 filter paper. The solvent from the crude extract were evaporated to air dried at room temperature.
Crude ethyl acetate extract (15g) was separated by silica gel (100-200 mesh) column (size 60cm x 4 cm) chromatography and eluted with hexane 100% followed by the combination of hexane : chloroform (9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8 and 1:9), then chloroform and Similarly the column was run over chloroform, then chloroform: ethyl acetate (9:1, 8:2 and 1:9) and then ethyl acetate respectively. A total of 118 fractions were collected in 10ml test tubes and pooled into 7 fractions based on similar RF values using thin layer chromatography.
2.3. Rearing of Test Insects
Egg mass of S. litura and different larval stages of H. armigera were collected from vegetable field at Anaipatti, Musiri, Trichirappalli, Tamil Nadu, and India. Larvae were reared in laboratory conditions (27.0˚C ± 2˚C; 70% RH) throughout the study period at PG & Research Department of Zoology, Government Arts College, Musiri, Tamil Nadu, India. Generally, healthy and uniform sized fourth instar larvae were used for the experiments and the cultures were maintained throughout the study period.
2.4. Antifeedant Activity
Antifeedant activity of the fractions of B.buxifolia was studied using leaf disc no choice method [10 ]. Required concentration of the fractions of B. buxifolia (1000ppm) was prepared by dissolving in acetone and mixing with dechlorinated water Polysorbate 20 (Tween 20) at 0.05% was used as an emulsifier [11 ]. Fresh cotton leaf (for H. armigera) and castor leaf (for S. litura) discs of 3 cm diameter were punched using a cork borer and dipped in 125,250, 500, and 1000ppm for fractions separately and air dried for 5 minutes. After air drying, treated leaf discs were kept inside the Petri dishes (15mm × 90 mm diameter) separately containing wet filter paper to avoid drying of the leaf disc and single 2hrs pre starved fourth instar larva of H. armigera and S. litura was introduced on each treated leaf disc.Neemazal was considered as constant. Ten replications were maintained for each treatment. A progressive consumption of leaf area by the larva in 24 hrs period was recorded in control and treatments using a leaf area meter (systronics 211). Leaf area consumed in plant extract and fraction treatments was corrected from the control. The percentage of antifeedant index was calculated using the formula of Ben Jannet, et al. [12 ].
C - T
AFI= -------------×100
C + T
Where
AFI = Antifeedant Index;
C = Area protected in control leaf disc;
T = Area protected in treated leaf disc.
2.5. Larvicidal Activity
For the evaluation of larvicidal activity of the fraction of B.buxifolia against the selected pest, primarily, the plant extract was tested on a wide range of concentration, from that a narrow range of concentration was derived. Thus, 125,250, 500, and 1000ppm concentrations for fractions were tested against the freshly moulted (0-6h) fourth instar larvae of H. armigera and S. litura .The branches bearing cotton leaves were tied with wet cotton plug to avoid early drying and placed in a plastic trough (29cm × 8cm). In each concentration 10 pre-starved (2hrs) fourth instar larvae were introduced individually and covered with muslin cloth. Neemazal was considered as constant. Five replicates were maintained for each concentration, each replicates comprised of 25 numbers of larvae. After 24h of the exposure period, the number of dead larvae was recorded from each replicates at all the concentrations and the percentage of larval mortality was calculated using Abbott’s formula [13 ]. The larvae with no symptom of a movement or shake while touching with soft camel brush were considered as dead.
%MT - %MC
Mortality (%) = --------------------------×100
100 - %MC
Where,
% MT = % Larvae mortality in treatment and
% MC = % Larvae mortality in control.
2.6. Ovicidal Activity
Twenty individual eggs of H. armigera andS.litura (for removal of scales from egg masses by using camel brush) were separated and dipped in various concentrations (as mentioned in antifeedant activity). Five replicates were maintained (n=100). Number of eggs hatched in the control and treatments were recorded and percent ovicidal activity was calculated according to Abbott [13 ] (as mentioned in larvicidal activity).
2.7. Statistical Analysis
Data analysis was carried out using Microsoft Excel 2007. One -Way ANOVA was performed for all the expe-rimental data from that Least Significant Difference was calculated and the significant differences were marked with different alphabet.LC50, LC90 was carried out using SPSS 16.00.
The results of the antifeedant potential of the solvent crude extracts of B. buxifolia investigated against S.litura and H. armigera larvae were presented in Table 1. Maximum antifeedat activity was recorded in fraction III followed by fractionVI against 74.33% and 57.32% for S. litura and 70.11% and 50.43% for H. armigera at 1000ppm concentration. Percentage ovicidal activity for fractions of B.buxifolia, studied at different concentration against S. litura and H. armigera was presented in table2. Maximum ovicidal activity was recorded in fraction III followed by fractionVI against 76.84% and 62.06% for S. litura and 73.12% and 67.02% for H. armigera at 1000ppm concentration. Percentage larvicidal activity for fractions of B.buxifolia, studied at different concentrations against S. litura and H. armigera was presented in table 3. Significantly promising larval mortality was recorded at 1000ppm concentrations of different fractions showed increased larvicidal activity in fraction fractionVI fraction III against (68.26% and78.66% for S. lituraand (58.84% and 73.76%) for H. armigera respectively.
The botanical extracts from the plant leaves, roots seeds, flowers and bark in their crude form have been used as conventional insecticides in throughout the world. Several authors have reported that plant extracts possess similar type of antifeedant, insecticidal, oviposition deterrent, ovicidal and growth inhibition activities against lepidopteran pests [14 ]. Antifeedant, larvicidal and insect growth inhibitory activities of Pseudocalymma alliaceum were studied against S. litura and H. armigera[15 ]. Antifeedant, larvicidal and insect growth inhibitory activities of Barleria longiflora were studied against S. litura and H. armigera[16 ]. Antifeedant, larvicidal and insect growth inhibitory activities of Pseudocalymma alliaceum were studied against S. lituraand H. armigera. Chinnamani, et al. [15 ]in the present study, it was observed that III fraction of B. buxifoliareduced the feeding rate of S. litura and H. armigera. Jeyasankar, et al. [17 ] reported that the possible insecticidal property in the selected plant may arrest the various metabolic activities of the larvae during the development and ultimately the larvae failed to moult and finally died. This is in accordance with the earlier findings of In the present investigation, III fraction of B. buxifolia at 1000ppm concentration was recorded then maximum larval mortality of 78.66% S. lituraand73.76% H. armigera. Secondary plant compounds act as insecticides by poisoning per se or by production of toxic molecules after ingestion. These compounds also deter or possibly repel an insect from feeding Lajide, et al. [18 ]. Baskar, et al. [19 ]Observed that twelve fractions were collected from hexane extracts of Couroupita guianensis were studied against H.armigera. Among them, eight fractions showed maximum percentage of larvicidal (80.88%) activity against H. armigera at 1,000ppm concentration respectively. In the present study III fraction isolated from ethyl acetate extract of B. Buxifolia exhibited statistically significant larvicidal activity against fourth instar larvae of S. lituraand H. armigera at 1000ppm concentrations. Present results agreed with Atalantia monophylla leaf extract was fractionated using silica gel column chromatography. Twelve fractions were collected and evaluated for their ovicidal activity at 125, 250, 500 and 1000 ppm concentrations. Among them, fraction 9 showed maximum ovicidal activity of 72.21% at 1000 ppm concentration with least LC 50 value of 435.92 ppm [20 ].
| Funding: The Authors are thankful that this work was conducted in the laboratory which is financially supported by UGC, New Delhi, India (Ref No. 42-570/2013 (SR)). |
| Competing Interests: The authors declare that they have no competing interests. |
| Contributors/Acknowledgement: The authors are thankful to Principal and Head of Department of Zoology, A. A. Govt. Arts College, Musiri-621 211, Tamil Nadu, India for their support and facilities provided. |
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Table-1.Antifeedant activity of ethyl acetate fractions of B.buxifoliaagainst fourth instars larvae of S.lituraand H.armigera
Fractions |
Spodopteralitura |
Helicoverpaarmigera |
||||||
Concentrations tested (ppm) |
||||||||
125 |
250 |
500 |
1000 |
125 |
250 |
500 |
1000 |
|
I |
7.56±3.08a (15.89) |
9.73±2.20a (18.15) |
10.38±2.22a (18.72) |
17.57±3.35a (24.73) |
8.25±3.64a (16.64) |
17.31±8.34bc (24.58) |
21.99±5.70b (27.90) |
26.35±7.49b (30.85) |
II |
9.18±2.38ab (17.56) |
19.24±2.19b (25.99) |
19.64±4.35bc (26.28) |
24.85±4.37b (29.87) |
9.75±5.75a (18.15) |
12.30±5.19ab (20.53) |
13.92±7.72a (21.89) |
21.16±3.88a (27.35) |
III |
21.10±4.02c (27.35) |
43.35±8.82c (41.15) |
59.42±7.31e (50.42) |
74.33±7.76e (59.54) |
23.73±4.73c (29.13) |
28.24±8.10c (32.08) |
43.99±7.41c (41.50) |
70.11±5.46e (56.85) |
IV |
6.34±2.52a (14.54) |
9.46±1.62a (17.85) |
13.37±1.65ab (21.39) |
17.05±4.71a (24.35) |
12.24±9.70b (20.44) |
14.30±6.34b (22.22) |
15.66±7.49ab (23.26) |
20.84±7.06a (27.13) |
V |
5.95±1.65a (14.06) |
13.06±4.54ab (21.13) |
22.35±6.26bc (28.18) |
28.76±8.39bc (32.39) |
6.36±3.56a (14.54) |
9.92±6.18a (18.34) |
11.51±5.26a (19.82) |
18.06±3.93a (25.10) |
VI |
17.18±3.35b (24.43) |
27.88±7.94c (31.82) |
40.36±6.61d (39.41) |
57.32±11.35d (49.20) |
14.29±2.97bc (22.14) |
17.16±2.22bc (24.43) |
21.19±4.41b (27.35) |
50.43±7.06d (45.23) |
VII |
8.83±2.75ab (17.26) |
14.18±7.08ab (22.06) |
16.45±4.78b (23.89) |
24.83±9.35b (29.87) |
7.65±3.82a (16.00) |
9.66±5.29a (18.05) |
12.76±3.47a (20.88) |
19.45±3.75a (26.13) |
Values are mean ±Standard deviation of five replications; Values in parentheses are angular transformed; ANOVA followed by Duncan Multiples Range Test (DMRT) was performed; Superscripts alphabet in the values are significantly different at p<0.05% Control group was fed with host plant without the treatment of chemicals.
Table-2.vicidal activity of ethyl acetate fractions of B.buxifoliaagainst fourth instars larvae of S.lituraand H.armigera
Fractions |
Concentration (ppm) |
S. litura |
H. armigera |
||||||
vicidal(%) |
LC50 |
LC90 |
X2 value |
Larvicidal (%) |
LC50 |
LC90 |
X2 value |
||
I |
125 250 500 1000 |
9.30±1.30a 10.10±0.46a 16.30±1.30a 27.10±3.49a |
1636.744 |
3031.141 |
0.571 |
7.20±1.35a 9.92±0.97a 12.60±1.43ab 17.10±2.77a |
1585.015 |
2821.249 |
0.915 |
II |
125 250 500 1000 |
10.42±1.13a 12.18±1.91a 18.90±3.57b 23.20±3.70ab |
1873.519 |
3567.500 |
2.463 |
10.84±0.70a 13.60±1.98ab 19.06±3.40bc 29.50±2.47b |
1508.466 |
2846.093 |
3.615 |
III |
125 250 500 1000 |
34.38±6.32d 48.20±4.65d 54.10±4.00d 76.84±3.87d |
437.466 |
1341.279 |
6.817 |
30.82±4.37c 46.14±2.77d 56.40±5.41e 73.12±2.48d |
465.191 |
1422.692 |
5.801 |
IV |
125 250 500 1000 |
9.60±2.30a 16.98±3.23b 20.54±3.90bc 28.42±4.46bc |
1617.994 |
3172.792 |
3.302 |
9.48±1.79a 16.14±3.06bc 20.50±1.39c 27.00±3.78bc |
1676.816 |
3272.828 |
3.412 |
V |
125 250 500 1000 |
7.88±1.86a 9.12±1.73a 10.50±1.87a 28.88±2.92a |
1569.626 |
2798.892 |
1.027 |
8.74±1.77a 9.42±0.98a 15.00±2.09a 26.40±3.65a |
1695.320 |
3129.784 |
0.189 |
VI |
125 250 500 1000 |
19.40±4.74c 26.02±4.18c 40.06±5.97c 62.06±2.37c |
744.960 |
1672.508 |
1.081 |
19.12±5.80b 28.42±4.99c 48.42±2.97d 67.02±3.19c |
1657.536 |
3174.053 |
1.136 |
VII |
125 250 500 1000 |
12.18±5.05b 16.16±4.11b 20.64±3.14bc 29.20±4.45b |
1689.712 |
3417.645 |
0.968 |
10.36±1.91a 13.88±2.05ab 16.88±2.58b 28.20±5.46b |
650.405 |
1472.027 |
4.322 |
Values are mean ± S.D of five replication; Number of larvae =10; LC50=Lethal concentration 50 and LC90=Lethal concentration 90; SPSS16.0. Values with different alphabet in column are statistically significant (p<0.05 level; DMRT).
Table-3.Larvicidalctivity of ethyl acetate fractions of B.buxifoliaagainst fourth instars larvae of S.lituraand H.armigera.
Fractions |
Concentration (ppm) |
S. litura |
H. armigera |
||||||
Larvicidal (%) |
LC50 |
LC90 |
X2 value |
Larvicidal (%) |
LC50 |
LC90 |
X2 value |
||
I |
125 250 500 1000 |
8.60±1.14a 12.80±2.41a 17.8±1.87a 28.48±1.46a |
1519.723 |
2791.434 |
3.127 |
8.90±2.84a 12.40±2.06b 18.20±2.58ab 26.70±4.40bc |
1620.130 |
3025.912 |
2.392 |
II |
125 250 500 1000 |
9.40±1.24a 16.40±2.51ab 20.30±1.63b 31.30±1.27ab |
1422.045 |
2699.516 |
4.918 |
15.90±2.57b 21.20±3.81bc 22.80±2.48b 28.40±2.99c |
1650.628 |
3346.041 |
3.759 |
III |
125 250 500 1000 |
21.80±1.85b 30.20±2.88d 51.20±7.09d 78.60±1.61d |
549.205 |
1198.750 |
4.317 |
27.10±4.52d 39.30±8.12d 51.30±3.10c 73.70±4.05d |
531.660 |
170.776 |
4.784 |
IV |
125 250 500 1000 |
9.40±2.07a 15.60±3.07a 16.50±3.78a 37.30±4.29a |
1264.146 |
2335.574 |
3.623 |
9.10±0.43a 10.30±0.54a 14.00±1.93a 26.80±3.22a |
1633.267 |
2238.010 |
1414 |
V |
125 250 500 1000 |
12.10±2.75ab 17.70±1.86b 18.10±2.26a 41.00±2.06a |
1185.616 |
2246.520 |
5.313 |
11.10±1.47ab 13.60±2.06a 16.80±4.54a 23.80±5.44b |
1616.643 |
3136.691 |
1.045 |
VI |
125 250 500 1000 |
21.60±1.20b 35.70±7.87c 47.10±2.87c 68.20±3.98c |
619.307 |
1494.693 |
4.715 |
20.80±1.08c 30.90±3.88c 50.70±5.65c 58.80±4.43c |
736.127 |
1767.499 |
5.290 |
VII |
125 250 500 1000 |
10.70±1.95a 15.80±2.80a 20.20±3.78ab 23.00±0.76b |
1961.831 |
3896.151 |
3.419 |
13.30±2.26a 19.40±2.41bc 24.20±6.25bc 31.30±4.98bc |
1561.350 |
3254.332 |
2.846 |
Values are mean ± S.D of five replication; Number of larvae =10; LC50=Lethal concentration 50 and LC90=Lethal concentration 90; SPSS16.0. Values with different alphabet in column are statistically significant (p<0.05 level; DMRT).