Bioefficacy of Couroupita guianensis (Aubl) against Helicoverpa armigera (Hub.) (Lepidoptera: Noctuidae) larvae

With a view to develop an ecofriendly botanical pesticide, we evaluated the bioeff icacy of crude extracts of Couroupita guianensis against third instar larvae of Helicoverpa armigera. Maximum feeding deterrency (81.67%) and least LC50 (2.72%) for larval mortality were seen in hexane extract. Hexane extract was subjected to column chromatography using different ratio of hexane-ethyl acetate solvent system. Totally eight fractions were collected. The fractions were screened at 125, 250, 500 and 1,000 mg kg concentrations against H. armigera using no-choice leaf disc method. Fraction eight showed maximum antifeedant (86.24%) and larvicidal (80.88%) activities at 1,000 mg kg concentration. C. guianensis could be utilized in pest control programme. Additional key words: antifeedant, hexane, larvicidal, phytochemicals.


Introduction
In the last five decades, many countries including India have concentrated on non-polluting and economic entomological technologies to increase the productivity of vegetable crops and economically important trees.Insect pests play a major role in damaging the crops and hence there is a need to use efficacious control agents.Crop loss due to insect pests varies between 10 to 30% for major crops (Ferry et al., 2004).In the last few decades, the repeated use of synthetic chemicals to manage these pests has lead to their resurgence and outbreak, resistance to insecticides, elimination of existing natural enemies and pollution of soil, water, air and food (Patel et al., 1992).Hence, search for viable and sustainable alternatives to synthetic pesticides is given priority (George and Seenivasagan 1998).More than 2,000 species of plants are known to possess insecticidal properties (Klocke, 1989).Prakash and Rao (1997) observed that plant pesticides do not contribute to resistance development or pest resurgence; nor do they cause negative effects on non-target organisms.They do not affect plant growth, seed viability and food quality but possess insecticidal, repellent, antifeedant and/or growth regulatory activities.Botanical pesticides tend to have broad-spectrum activity, are relatively specific in their mode of action, easy to process, produce, use and safe for higher animals and the environment (Talukder and Howse, 1994).Plants are endowed with a potential to produce a range of secondary metabolites like alkaloids, terpenoids, flavonoids, phenols, glycosides, sitosterols and tannins.These phytochemicals are known to protect the plants from the attack of insect-pests (Ahmad, 2007).The phytochemicals produced in response to insect-pest attack, affect feeding and oviposition of insects on the plants.Phytochemicals have shown different kinds of actions that can modify specific physiological processes in insects (Liu et al., 2007).Koul et al. (2003) noticed that plant chemicals' defense against insect herbivory almost never depends on a single compound, but instead several compounds interact with pests, individually or in unison.Application of biopesticides has been reported to have positive impacts on bollworm population management (Ge and Ding, 1996).Most of the tested botanical extracts proved to be strong growth inhibitors, acutely toxic and active feeding deterrents against lepidopteran species (Akhtar et al., 2008).The American bollworm, Helicoverpa armigera is a polyphagous noctuid feeding on 181 plant species of 39 families.Couroupita guianensis (Aubl.)belonging to the family Lecythidaceae possesses insecticidal properties.Methanol extract derived fractions of C. guianensis inhibited the growth of microorganisms (Khan et al., 2003).Petroleum ether and chloroform extracts of this plant exhibited larvicidal activity against vectors (Desal et al., 2003).The present study was undertaken to assess the antifeedant and larvicidal activities of C. guianensis against the notorious agricultural pest, H. armigera.

Plant collection and extract preparation
Leaves of C. guianensis were collected from Loyola College Campus, Chennai, Tamil Nadu, India, on 25 th October 2005.The plant was identified by Dr. Ayyanar, taxonomist, at Entomology Research Institute, Loyola College.The voucher specimen (ERIH: 1310) was deposited at the institute herbarium.The plant material was shade dried at room temperature and powdered coarsely.The powder (2.0 kg) was soaked in hexane, chloroform and ethyl acetate for a period of 48 h sequentially with intermittent shaking and filtered.The extracts were concentrated under reduced pressure using rotary evaporator and stored at 4°C.The yield of hexane extract was 23.4 g, chloroform extract was 20.9 g and ethyl acetate extract was 22.1 g.

Phytochemical screening
Phytochemical analysis of the crude extracts was carried out following the method of Harbone (1998).

Insect culture
Larvae of H. armigera were collected from the field in Salamangalam, Kancheepuram district, Tamil Nadu.The collected larvae were reared individually in a plastic container (vials) and fed regularly with bhendi, Abelmoschus esculentus L. (Malvaceae) till the larvae became pupae under the laboratory conditions (27 ± 2°C and 75 ± 5% relative humidity).Sterilized soil was provided for pupation.After pupation, the pupae were collected from soil and placed inside the cage for emergence of adults.Cotton soaked with 10% honey solution mixed with a few drops of multivitamins was provided for adult feeding to increase the fecundity.Potted cowpea plant was kept inside adult emergence cage for egg laying.After hatching the larvae were collected from the cage and fed with standard artificial diet (Koul et al., 1997).The laboratory reared third instar larvae were used for the present investigation.

Antifeedant activity
Antifeedant activity of the crude extracts was studied using leaf disc no-choice method.Fresh cotton leaf discs of 4 cm diameter were punched using cork borer.They were dipped in 0.5%, 1.0%, 2.5% and 5.0% concentrations of crude extracts and 125, 250, 500 and 1,000 mg kg -1 concentrations of fractions individually.The leaf discs dipped in acetone were used as negative control since acetone was used to dissolve the crude extracts and fractions.Azadirachtin (40.86% purity, obtained from EID-parry, India Ltd., Chennai) was used as positive control.In each plastic Petri dish (1.5 cm × 9 cm) wet filter paper was placed to avoid early drying of the leaf discs and single third instar larva was introduced into each Petri dish.Progressive consumption of leaf by the treated and control larvae in 24 h was recorded using Leaf Area Meter (Delta-T Devices, Serial No. 15736 F 96, U.K). Leaf area eaten by larvae in treatment was corrected from the negative control.Five replicates were maintained for each treatment with 10 larvae per replicate (total, n = 50).The experiment was conducted at laboratory condition (27 ± 2°C) with 14:10 light and dark photoperiod and 75 ± 5% relative humidity.Antifeedant activity was calculated according to the formula of Bentley et al. (1984): Leaf area consumed in control -leaf area consumed in treated leaf Antifeedant activity = -------------100 Leaf area consumed in control

Larvicidal activity
Larvicidal activity was studied using leaf disc nochoice method.The cotton leaf discs were dipped in different concentrations of crude extracts and fractions.They were placed in petri dishes and the larvae were introduced as in the antifeedant experiment.After 24 h treatment the larvae were continuously maintained on the nontreated fresh cotton leaves.Diet was changed every 24 h.Larval mortality was recorded after 96 h of treatment.Five replicates were maintained for each treatment with 10 larvae per replicate (total n = 50).The laboratory conditions were the same as in the antifeedant experiment.Percent mortality was calculated according to Abbott (1925): % mortality in treatment -% mortality in control Abbott corrected mortality = ----------100 100 -% mortality in control

Statistical analysis
The data related to antifeedant and larvicidal activities were analysed using one way ANOVA.Signif icant differences between treatments were determined using Tukey's multiple range test (P ≤ 0.05).LC 50 and LC 90 values were calculated using probit analysis (Finney, 1971).

Phytochemical analysis
Phytochemical investigation of the crude extracts revealed the presence of alkaloids, coumarin and quinone in the crude extracts of C. guianensis (Table 1).

Antifeedant activity
The present study revealed that maximum antifeedant activity of 81.67% was recorded in hexane leaf extract of C. guianensis at 5.0% concentration followed by chloroform (73.68%) and ethyl acetate (69.70%) extracts (Table 2).The effective hexane crude extract was fractionated using silica gel column chromatography with different solvent system; finally eight fractions were isolated and they were screened for antifeedant activity.Fraction 8 showed highest antifeedant activity at all concentrations tested.Maximum antifeedant activity of 86.24% was noticed in fraction 8 at 1,000 mg kg -1 concentration (Table 3).Third and seventh fractions recorded more than 70% of feeding deterrency at 1,000 mg kg -1 concentration.Minimum antifeedant activity was recorded in fourth fraction.

Larval mortality
Toxic effect was observed in all the extracts.Maximum effect was seen in hexane extract at 5.0% concentration with the LC 50 value of 2.72% followed by chloroform and ethyl acetate extracts with LC 50 values of 5.21 and 7.22% respectively (Table 4).The Chi-square values were significant at P < 0.05 level.The high Chisquare values in the bioassays probably indicated the heterogeneity of the test population.Different fractions influenced larval mortality differently.Maximum larval mortality of 80.88% was observed in eighth fraction at 1,000 mg kg -1 and the LC 50 and LC 90 values were 413.30 and 1,181.68mg kg -1 respectively (Table 5).Fifth fraction showed the LC 50 and LC 90 values at 1,456.98 and 2,233.87mg kg -1 concentrations respectively.Larval mortality was nil in fourth fraction.Third, seventh and eighth fractions showed larvicidal activity at all concentrations.Less than 50% of larval mortality was observed in fifth and sixth fractions.Statistically significant activity was seen in fraction 8 when compared with other fractions.Low polar hexane extract and fractions of hexane extract produced high larval mortality comparable to that of Azadirachtin.

Discussion
In the present study maximum antifeedant activity was observed in hexane extract.The antifeedant activity was high in the low polar hexane extract.Tewary et al. (2005) reported that low polar solvent extracts had higher activity than high polar solvent extracts.Morimoto et al. (2002) reported that hexane extract of Galium aparine had higher phagodeterrent activity against Spodoptera litura.Similarly hexane extracts from Emblica officinalis and Ocimum sanctum, showed good antifeedant activity against S. litura (Sharma and Bisht, 2008).Also, hexane portion from methanol extract of Cryptomeria japonica showed higher antifeedant activity against Locusta migratoria (Wu et al., 2008).Eighth fraction showed higher antifeedant activity against H. armigera in our study.Raja et al. (2005) reported that second fraction obtained from ethyl acetate extract of Hyptis suaveolens eluted with hexane showed potent antifeedant activity against H. armigera.
Phytochemical analysis revealed the presence of alkaloids, coumarin and quinone in the hexane extract of C. guianensis which probably contributed to the feeding deterrency in the present study.Verma et al. (1986) reported that alkaloids present in plants inhibited the feeding of S. litura.Feeding behavioral experiments showed that alkaloids acted as potent feeding deterrents in lepidopteran larvae (González-Coloma et al., 2004;Kathuria and Kaushik, 2005).Quinone was known to possess antifeedant activity against a variety of agricultural pests (Krishnakumari et al., 2001).Coumarin and quinone acted as feeding deterrents against S. litura (Morimoto et al., 1999).In the present study higher mortality was recorded due to the presence of alkaloids, coumarin and quinone.The results of the present investigation corroborate with the findings of Baskar et al. (2009) who reported that presence of alkaloids, coumarin and quinone present in the hexane extract of Atalantia monophylla showed higher larval mortality in H. armigera.Akhtar et al. (2008) reported that alkaloids acted as neuro-muscular toxin, contact and stomach poison.Coumarin acted as insecticide against four different insect pests (Moreira et al., 2007).Georges et al. (2008) reported that anthroquinone exhibited insecticidal activity.
Fresh cotton leaf treated with hexane extract and its fractions recorded minimum consumption which lead to death of H. armigera larvae.This finding coincides  (Subashini et al., 2004).Malarvannan et al. (2009) observed that hexane extract from Clausena dentata showed 100% ovicidal activity against H. armigera.Khalequzzaman and Sultana (2006) reported that petroleum extract of Annona squamosa seeds recorded higher larval mortality than ethyl acetate and acetone extracts.Petroleum ether extract of Synedrella nodiflora caused 80% larval mortality in S. litura (Martin Rathi and Gopalakrishnan, 2006).
The present study will be useful in promoting the development of new botanical insecticide to manage herbivores.

Table 2 .
Per cent antifeedant activity of crude extracts of C. guianensis against H. armigera

Table 3 .
Antifeedant activity (%) of C. guianensis fractions against H. armigera a Within the column, means ± SD followed by the same letter does not differ significantly (Tukey's test, P ≤ 0.05).

Table 5 .
Aravinda et al. (2009)) LC 50 -LC 90 and χ 2 values of C. guianensis fractions against H. armigera Within the column, means followed by the same letter do not differ significantly (Tukey's test, P ≤ 0.05).*χ 2 values are significant at P < 0.05 levels.FL: fiducial limit.with the observations ofKamaraj et al. (2008)who studied the extract of Ocimum canum, O. sanctum and Rhinacanthus nasutus treated leaf discs.They noticed low consumption with high mortality in the larvae of H. armigera.Similar findings were observed in low polar solvent system in different plant extracts against H. armigera.Janarthan et al. (1999)reported that petroleum ether extracts of Parthenium histerophorus at a concentration of 0.2 and 0.5% caused 100% mortality in the larvae of H. armigera.Aravinda et al. (2009)found that different solvent (petroleum ether, hexane) extracts of Jatropha curcas showed more than 70% insecticidal activity against larvae of H. armigera.Hexane extract of Dodonaea angustifolia reduced the fecundity, hatchability and larval mortality of H. armigera