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Chilean journal of agricultural research

On-line version ISSN 0718-5839

Chilean J. Agric. Res. vol.72 no.1 Chillán Mar. 2012

http://dx.doi.org/10.4067/S0718-58392012000100009 

Chilean Journal of Agricultural Research 72(1) January - March

RESEARCH

Insecticidal Efficacy of Azadirachta indica, Nucleopolyhedrovirus and Chlorantraniliprole Singly or Combined against Field Populations fo Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Eficacia Insecticida de Azadirachta indica, Nucleopolihedrovirus y Clorantraniliprol solo y sus Aplicaciones Integradas contra Poblaciones de Campo de Helicoverpa armigera Hübner (Lepidoptera: Noctuidae)

Waqas Wakil1*, M. Usman Ghazanfar2, Fozia Nasir1, Mirza Abdul Qayyum1, and Muhammad Tahir1

1University of Agriculture, Department of Agricultural Entomology, Faisalabad, Pakistan."Corresponding author (waqaswakeel@hotmail.com, arid1972@yahoo.com).
2University College of Agriculture, University of Sargodha, Department of Plant Pathology, Sargodha, Pakistan.


The development of resistance in cosmopolitan insect Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) forced the researchers for alternative control measures. In the present study, insecticidal efficacy of formulations of Azadirachta indica, a Nucleopolyhedrovirus (NPV), and new anthranilic diamide insecticide (chlorantraniliprole) formulations was determined against 2nd, through 5th larval instars of H. armigera collected from diverse geographical locations in the Punjab province, Pakistan. Azadirachta indica was applied at 5 μL L-1; NPV at 2.1 x 105 polyhedral occlusion bodies (POB) mL4 and chlorantraniliprole at 0.01 μL L-1, either alone or in combinations with each other. The bioassays were conducted at 27 ± 1 °C and 65 ± 5% relative humidity. The mortality varied greatly among treatments, larval instars, and locations. The combinations of NPV with A. indica and chlorantraniliprole caused higher mortality, pupation and produced an additive effect compared to their application singly in all the tested populations. The population from Rawalpindi was always susceptible while the Gujranwala was the resistant. The results herein suggest that the effectiveness of NPV and A. indica can be improved by the presence of chlorantraniliprole against the larvae of H. armigera.

Key words: Azadirachta indica, NPV, chlorantraniliprole, additive, Helicoverpa armigera, populations.


Se determinó la eficacia insecticida de formulaciones de Azadirachta indica, Nucleopolihedrovirus (VPN) y el nuevo insecticida diamida antranílico (clorantraniliprol) en contra de segundo, tercero, cuarto y quinto estadios larvales de Helicoverpa armigera Hubner (Lepidoptera: Noctuidae) recogidos de diversas ubicaciones geográficas de la provincia de Punjab, Pakistán. Azadirachta indica se aplicó en dosis de 5 μL L-1; VPN en dosis 2.1 x 105 POB mL-1 y clorantraniliprol fue 0,01 μL L-1 ya sea solos o en combinaciones. Los bioensayos se realizaron a 27 ± 1 °C y 65 ± 5% de humedad relativa. La mortalidad fue notablemente variada entre los tratamientos, estadios larvales y diversas poblaciones. Las combinaciones de VPN con A. indica y clorantraniliprol dio una mayor mortalidad, la fase de pupa y el efecto aditivo producido en comparación con su aplicación solo en todas las poblaciones de la prueba. La población de Rawalpindi fue siempre susceptible, mientras que Gujranwala fue resistente. Los resultados del presente trabajo sugieren que la eficacia de VPN y A. indica pueden ser beneficiados por la presencia de clorantraniliprol contra las larvas de H. armigera.

Palabras clave: Azadirachta indica, VPN, clorantraniliprol, aditivos, Helicoverpa armigera, poblaicones.


Helicoverpa armigera Hübner (Lepidoptera: Noctuidae), a major polyphagous insect pest of many crops (Marzban et al., 2009), has a wide geographical range, mobility, migratory potential, facultative diapause, high fecundity and a great tendency to develop resistance against insecticides, key factors that determine its importance as insect pest (Fitt, 1989; Zalucki, 1991; Wakil et al., 2009a; 2009b; 2010). Excessive use of synthetic insecticides worldwide warrants environmental and human health concerns, and urges researchers to develop safer alternatives for eco-friendly pest management (Cherry et al., 1997). Insect resistance to synthetic insecticides (Ahmad et al., 2003; 2007) and development of awareness of their detrimental effects has prompted the introduction of integrated pest management programs (Nathan and Kalaivani, 2006). The promising alternatives of insecticides would be Nucleopolyhedrovirus (NPV), plant based products and new chemistry molecules which can be successfully included in the integrated pest management (IPM) program to lessen the resistance issues in the lepidopterous insects.

Helicoverpa armigera, single nucleocapsid NPV (HearNPV, also called HaSNPV) was first isolated in 1975 in Hubei, China and has been used for over 25 yr against H. armigera (Zhang, 1994). The Baculoviridae including NPVs and granuloviruses (GV) with more than 600 viruses (Nathan and Kalaivani, 2006) have great potential for the control of H. armigera (Jayaraj, 1985; David, 2008). Occluded viruses (OV) initiate primary infections in midgut epithelial cells of susceptible hosts and Budded viruses (BV) spread from cell to cell in the larvae (Keddie et al., 1989; Washburn et al., 1995). The infected larvae become pale in color, which ultimately swell due to the deposition of OV's, climb upper parts of the host plants and ultimately die (Inceoglu et al., 2001; Nakai et al., 2002).

Over the last three decades, Azadirachta indica A. Juss (neem) has received attention all over the world (Stark and Walter, 1995; Nathan and Kalaivani, 2006). The major constituent of neem is azadirachtin (AZA), which affects the feeding, growth, molting, and reproduction of insects (Kumar et al., 2008), and may be combined with other bio-based insecticides (Koppenhòfer and Kaya, 2000). The effects of neem based pesticides have been studied under laboratory and field conditions (Gahukar, 1995; 1996), however, in Pakistan the efficacy of the neem has not been properly explored against H. armigera except few studies like Wakil et al. (2008).

The chlorantraniliprole (Rynaxypyr) is the first anthranilic diamide, a new class of insecticide (Lahm et al., 2007) that prevents the build-up of pest populations if applied soon after pest outbreaks. It has very low mammalian toxicity, acts at relatively low application rates, promotes eco-friendly insect pests management and achieves excellent control of pest population resistant to other insecticides (Cordova et al., 2007). It activates the insect ryanodine receptors (RyRs), which causes uncontrolled release and depletion of internal Ca and prevents further muscle contraction. The death of insects occurs by the rapid cessation of feeding, lethargy, regurgitation and muscle paralysis (Cordova et al., 2007; Temple et al., 2009).

This research was aimed to evaluate mortality and pupation rate of A. indica and NPV in combination with chlorantraniliprole against 2nd, through 5th larval instars of H. armigera from several locations in the Punjab province, Pakistan.

MATERIALS AND METHODS

Insects rearing
Field populations of H. armigera were collected in six major tomato (Solanum lycopersicum L.) growing locations (Faisalabad, Gujranwala, Lahore, Rawalpindi, Sargodha, Sheikhupura) in Punjab, Pakistan. All the populations were reared in Integrated Pest Management (IPM) laboratory in the Department of Agricultural Entomology, University of Agriculture Faisalabad (Pakistan) at 25 ± 2 °C, 75% RH and 16:8 h photoperiod. Batches of 500 field collected larvae of each population were reared in plastic trays with 32 wells (6 cm diameter x 5.5 cm depth) with one larva each to avoid cannibalism, and provided with 5 mL of artificial diet (chickpea [Cicer arietinum L.] flour: 125 g; red kidney beans [Phaseolus vulgaris L.]: 125 g; canned tomotao paste: 25 g; agar: 17 g; ascorbic acid: 3 g; sorbic acid: 1 g; yeast: 40 g; methyl-4-hydroxybenzoate: 2 g; streptomycin: 1 g; vitamin mixture: 10 mL; distilled water: 1300 mL) (Wakil et al., 2011), which was renewed regularly till pupation. Twenty five pairs of unsexed newly emerged adults of H. armigera were placed in plastic jars (15 cm diameter x 19 cm depth) lined with coarse tissue paper to facilitate egg laying. Honey solution (10%) was provided in 5 mL test tube plugged with cotton, placed vertically on the top of each jar. Populations were reared for more than 10 generations in the laboratory before the bioassays, where L2 through L5 larval instars were used.

Test formulations
Azadirachta indica. A commercial product of neem A. indica (AgriLife, Hyderabad, India) was used at the rate of 5 μL L-1.

Chlorantraniliprole. A novel insecticide from class anthranilic diamide Coragen 20% SC powered by Rynaxypyr (DuPon Private Limited, Pakistan) was used which contains chlorantraniliprole (20% w/v) and other ingredients (80% w/v). The formulation was applied by dissolving in distilled water at a concentration of 0.01 μL L-1 mixed with the diet, thoroughly mixed in electric shaker for 30 s for even distribution of insecticide.

Nuclear Polyhedrosis Virus (NPV). The commercial formulation of NPV was provided by AgriLife, Hyderabad, India. To recover the purified virus, the second instar larvae of H. armigera were infected with a suspension of NPV by spraying and the larvae were allowed to feed on artificial diet (Wakil et al., 2011) for 7 d. Then the midguts of the infected cadavers were homogenized in deionized water, filtered through muslin cloth and centrifuged at 16 000 rpm for 45 min (Shapiro et al., 2005; Green et al., 2006). The purified virus was given washes three times in distilled water, and held in 0.1 mM NaOH at 5 °C. Polyhedral occlusion bodies (POB) in 1 mL suspension was recorded 10 times using haemocytometer (Cory and Myers, 2004). The formulation was applied (2.1 x 105 POB mL-1) on both sides of surface sterilized tomato leaf discs and air dried.

Larval treatment
Pre-starved (24 h) larval instars (L2-L5) from all populations of H. armigera were kept in plastic vials (base radius 2.8 cm x height 7 cm) individually containing tomato leaf discs treated with NPV and A. indica, alone and in combination while the discs sprayed with distilled water only served as control. After 24 h the larvae from alone treatments were removed and shifted into new vials (base radius 2.8 cm x height 7 cm) each containing 1 cm3 pieces from chlorantraniliprole treated and untreated (control) diet for next 24 h and then were shifted to normal artificial diet. For alone treatments (NPV and A. indica and chlorantraniliprole) the larvae were held in the vials separately for 24 h and directly transferred to the artificial diet.

Bioassay
The bioassay was conducted at 25 ± 2 °C, 75% RH and 16:8 h photoperiod, each treatment was repeated thrice independently using 20 larvae per replicate for each population (n = 360 for all populations). The mortality counts were made after every other day upto 12 d for all populations and larval instars (L2-L5). After removing the dead individuals the remaining larvae were kept till pupation. The larvae were prodded with the blunt needle and those unable to move in coordinated manner were considered as dead (Ma et al., 2008). The larvae infected with NPV showed the symptom of oozing of body contents, transparency and stretching of the body, A. indica application resulted in blackening and shrinkage of the larval body, however, the combined treatments resulted in the larval blackening and oozing of the body contents (Kumar et al., 2008).

Statistical analysis
Data were analyzed statistically by using three-way factorial analysis (Minitab, 2003) both for the mortality and pupation rate for six localities, four larval duration and seven treatments. The means of corrected percent mortality and pupation rate was separated and compared using Tukey-Kramer (HSD) (Sokal and Rohlf, 1995) test at 5% significance level. The type of interaction between different concentrations for the H. armigera mortality was worked out by using the equation CTF = (oc-oe)/oe x 100, where CTF is cotoxicity factor, oc is observed percentage mortality resulted from the combined application and oe, the expected percentage mortality, is the sum of percentage produced by each of the treatment used in the combination (Mansour et al., 1966). On the basis of this factor, any intermediate value (i.e. between -20 and +20) was considered additive (Marzban et al., 2009).

RESULTS

All main effects and the interaction between larvae and treatments were significant, however, other associated interactions at P = 0.05 were not significant both for mortality and pupation rate (Table 1) of H. armigera larvae. The mortality of larvae and pupation was greatly influenced by the origin of the population and the stage of the larvae as with the advancement of growth, the larvae became more resistant to the treatments. Significant differences were noted in the mortality and pupation rate of second instar larvae of H. armigera when exposed to chlorantraniliprole, NPV, and A. indica alone and in combinations. The additive effect on the mortality and pupation of H. armigera was exhibited by the combined treatments of chlorantraniliprole, NPV, and A. indica. The highest mortality was recorded in the treatment where NPV and chlorantraniliprole was applied in combination, with lowest pupation of H. armigera, in all the populations. The population from Gujranwala showed resistance with 96.10% mortality and the pupation was 0.13% while the population from Rawalpindi exhibited susceptibility towards the combined treatment with 100% mortality with no pupation (Table 2). However, in case of larvae treated with chlorantraniliprole alone, significantly more mortality was recorded in all the populations tested with maximum 63.23% and 33.10% pupation in Rawalpindi populationbut this was less effective in Gujranwala population with 51.23% mortality and 45.13% pupation.

Table 1. Factorial analyis of localities, larval duration and treatments on the mortality and pupation of 2nd, 3rd, 4th and 5th larval instars of Helicoverpa armigera from six different localities treated with Azadirachta indica, Nucleopolyhedrovirus (NPV), and chlorantraniliprole.

Table 2. Mean mortality and pupation of 2nd instar larvae of Helicoverpa armigera from six different locations treated with Azadirachta indica (Ai: 5 μL L-1), Nucleopolyhedrovirus (NPV: 2.1 X 105 POB mL-1) and chlorantraniliprole (Ch: 0.01 μL L-1) alone and in combination.

Percentage means within the locality followed by the same letter are not significantly different, Tukey-Kramer HSD test at P < 0.05. POB: Polyhedral occlusion bodies, CTF: Cotoxicity factor.

 

The mortality in 3rd instar larvae of H. armigera was higher when exposed to the combined treatments of chlorantraniliprole, NPV, and A. indica with less pupation respectively in all the populations. The larval mortality was significantly higher by the combination of NPV with chlorantraniliprole than chlorantraniliprole alone. Also the interaction between the combined treatments was additive depending upon the origin of the population. The population of Gujranwala showed resistance with 91.11% mortality and 3.89% pupation while the population of Rawalpindi exhibited susceptibility towards the combined treatment with 100% mortality with no pupation (Table 3). However, in case of larvae treated with chlorantraniliprole alone, significantly more mortality was recorded in all the populations tested with maximum mortality of 60.40% and 35.28% pupation in Rawalpindi population but this was less effective in Gujranwala population (47.13% mortality and 49.43% pupation).

Table 3. Mean mortality and pupation of 3rd instar larvae of Helicoverpa armigera from six different locations treated with Azadirachta indica (Ai: 5 μL L1), Nucleopolyhedrovirus (NPV: 2.1 X 105 POB mL1) and chlorantraniliprole (Ch: 0.01 μL L1) alone and in combination.


Percentage means within the locality followed by the same letter are not significantly different, Tukey-Kramer HSD test at P < 0.05. POB: Polyhedral occlusion bodies, CTF: Cotoxicity factor


The same trend was observed in case of mortality in 4th instar larvae of H. armigera which was higher when exposed to the combined treatments of chlorantraniliprole, NPV, and A. indica with less pupation respectively in all the populations. The larval mortality was significantly increased by the combination of NPV with chlorantraniliprole over that in chlorantraniliprole alone. Similarly the interaction between the combined treatments was additive depending upon the origin of the population. The population of Gujranwala was more resistant with 58.07% mortality and the 36.56% pupation, whereas the population of Rawalpindi exhibited susceptibility towards the combined treatments with 77.83% mortality and 16.90% pupation (Table 4). However, in case of larvae treated with chlorantraniliprole alone, significantly higher mortality was recorded in all the populations tested with maximum 41.79% and 52.97% pupation in Rawalpindi population but this was less effective in Gujranwala population with 29.55% mortality and 65.08% pupation.

Table 4. Mean mortality and pupation of 4th instar larvae of Helicoverpa armigera from six different locations treated with Azadirachta indica (Ai: 5 μL L-1), Nucleopolyhedrovirus (NPV: 2.1 X 105 POB mL-1) and chlorantraniliprole (Ch: 0.01 μL L-1) alone and in combination.

Percentage means within the locality followed by the same letter are not significantly different, Tukey-Kramer HSD test at P < 0.05. POB: Polyhedral occlusion bodies, CTF: Cotoxicity factor.

 

The additive effect was noted in 5th instar larvae by the combined treatments of chlorantraniliprole, NPV, and A. indica among all the populations tested and mortality was higher in the combined treatments than the individual treatments (Table 5). The decreasing order of susceptibility at NPV and chlorantraniliprole treatment was Rawalpindi population (mortality 67.81%, pupation 28.94%), Sargodha (65.64%, 31.06%), Lahore (62.29, 32.45), Faisalabad (60.41, 36.27), Sheikhupura (57.46, 39.29) and Gujranwala (54.40, 42.33).

Table 5. Mean mortality and pupation of 5th instar larvae of Helicoverpa armigera from six different locations treated with Azadirachta indica (Ai: 5 μL L1), Nucleopolyhedrovirus (NPV: 2.1 X 105 POB mL-1) and chlorantraniliprole (Ch: 0.01 μL L-1) alone and in combination.

Percentage means within the locality followed by the same letter are not significantly different, Tukey-Kramer HSD test at P < 0.05. POB: Polyhedral occlusion bodies, CTF: Cotoxicity factor.

 

DISCUSSION

Long term use of synthetic insecticides have culminated into serious health and environmental issues (Nathan and Kalaivani, 2006) which redirect the researchers to look for some safer alternatives and ecologically acceptable pesticides (Wood and Granados, 1991) with no or less residual effect and resistance development property, for the control of important insect pests. The results of the present study indicate that NPV, A. indica, and chlorantraniliprole can be used successfully against various larval instars of H. armigera, but their effectiveness may depend on several factors like the origin of the populations and the stages of the larvae. Decline in susceptibility of H. virescens to NPV, azadirachtin, and imidacloprid has been documented after second instar by Koppenhofer and Kaya (2000). Kumar et al. (2008) supported the earlier notion about the susceptibility of the early instar larvae compared to the late instars as they ingest more treated leaf area during scrap feeding. However, in another study against Spodoptera litura F. (Murugan et al., 1999) the maximum mortality of larval stages was observed at highest concentrations of virus but Allen and Ignoffo (1969) noted that the susceptibility decreases with the age of larvae. These results are in confirmation to the present work as the age of the H. armigera larvae increased the mortality of the larvae decreased. The reason of this behavior is not yet explored properly except Nathan et al. (2005), who studied the effects of azadirachtin (AZA) and NPV on midgut enzyme activity in Spodoptera litura and noted that the gut enzyme activities were decreased by AZA and NPV individually and in combination. Upon feeding on diet of castor leaves treated with AZA and NPV in bioassays, gut enzyme-acid phosphatases, alkaline phosphatases, adenosine triphosphatases, and lactate dehydrogenase-activities decreased in S. litura early instar larvae. But still there is room for more research on the biochemical, molecular, and histopathological studies of the midgut necessary to understand the mechanism of decreased susceptibility of aging larvae.

Presently, the viral formulations (especially belonging to the family Baculoviridae) are one of the most promising biological insecticides (Lavina et al., 2001). Nuclear Polyhedrosis Viruses move from cell to cell and after its ingestion, resulted infected cells disintegrate which ultimately leads to the death of the insects. While, the neem has great antifeedant, insect growth regulatory and insecticidal effects, containing several compounds (Ramya and Jayakumararaj, 2009). The principal active ingredient in neem is AZA, which disrupt the action of molting hormone, reproduction of insects (Lee et al., 1991) and can be mixed with other biopesticides, microbials or with synergists (Koppenhòfer and Kaya, 2000). There are reports regarding the interaction of NPV and AZA against other insects with varying success. Shapiro et al. (1994) found that addition of neem extract with NPV against gypsy moth resulted in faster mortality, similarly, Nathan and Kalaivani (2006) evaluated NPV with AZA against S. litura and noted dose and larval instar dependent growth retardation after treatment with NPV and AZA and also decrease in nutritional indices by two fold at low concentrations. Nathan et al. (2005) exhibited the synergistic effect of botanical insecticides and virus when combined in low doses. The above narrated results are in confirmation with our findings as combinations are more effective in reducing the populations of H. armigera which also showed the additive effect compared to alone treatments. Cook et al. (1996) also endorsed these findings when they treated second instar gypsy moth larvae with AZA and NPV that resulted in higher mortality compared with that caused by individual treatments so both AZA and virus in combination may give good foliage protection against gypsy moth larvae. The synergistic action of AZA and NPV was also observed by Nathan and Kalaivani (2005) against tobacco cutworm and this interaction was not dose dependent.

Our findings suggest that presence of chlorantraniliprole enhanced the insecticidal activity of NPV and A. indica as the present study is the first report on potential combination of all three agents against the larvae of various populations of H. armigera. Less mammalian toxicity, effectiveness at relatively low dose rates as compared to other insecticides (pyrethroids, organophosphates, and carbamates), longer residual properties, and wide range of activity of chlorantraniliprole against lepidopteran pests will make it as an excellent control option in an overall integrated pest management system (Anonymous, 2007). It demonstrated very good activity at relatively low rates against larvae and pupae evaluated in this study and insecticidal activity of chlorantraniliprole was at par with that reported by Lahm et al. (2007). In laboratory study, LC50's for chlorantraniliprole (0.1 μL L-1) have been significantly lower when compared with other two standard insecticides indoxacarb (1.5 μL L-1) and cypermethrin (13.5 μL L-1) in an insecticide-treated diet assay on a laboratory colony of tobacco budworm (Anonymous, 2007).

The variation in the susceptibility levels among insecticides is likely to occur among populations originating from different parts of the world or even from the same region. Our study is the first in which variable effectiveness of NPV, A. indica, and chlorantraniliprole against H. armigera populations originating from different locations in the Punjab province (Pakistan) is documented. In the light of our findings, the population from Gujranwala appeared to be more tolerant to NPV and A. indica and chlorantraniliprole compared with the remaining populations collected from other localities. Resistance to different conventional insecticides has been observed in varying degree in lepidopteran insect populations (Ahmad et al., 2003; 2007), which might be attributed to the excessive applications of insecticides on different field crops grown in that particular localities.

CONCLUSIONS

Among various evaluated treatments, NPV+Ch proved to be the best one against all the populations of H. armigera further 2nd stage larvae were the most susceptible one. Present laboratory study suggests that, under specific experimental conditions, NPV and A. indica can be used with success in conjunction with chlorantraniliprole against various larval instars of H. armigera, thereby allowing a lower dose of their combination for the management of H. armigera. Findings of the present study earnestly suggest that the mortality demonstrated in the laboratory does not give the true picture of the mortality in the field and also the generalizations should be avoided. More research is needed to have better idea on the interaction of chlorantraniliprole, NPV, and A. indica to control the H. armigera both in laboratory and field conditions. The data generated here comprises initial efforts in establishing baseline information about chlorantraniliprole that can be used as reference points for future integration with other safer microbial based management programs.

ACKNOWLEDGEMENTS

The study was financially supported by grant (N° 1185) from Higher Education Commission (HEC), Islamabad, Pakistan, under National Research Program for Universities.

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Received: 26 June 2011.
Accepted: 27 December 2011.