Background

The Oriental leaf worm moth, Spodoptera litura (F.) (Lepidoptera: Noctuidae) is a serious polyphagous and cosmopolitan insect pest of cash crops, vegetable and ornamentals (Senthil-Nathan and Kalaivani, 2005). In Pakistan, S. litura causes heavy losses in various regions such as the northern and southern districts of Punjab (Ahmad et al. 2007). The increase in area under cultivation of succulent crops like soybean, cotton, mung bean, cabbage, and vegetables provide ideal conditions for its vigorous reproduction, resulting in a rapid increase of generations and population size (Gao et al. 2004). Mainstay of the farming community to combat this pest is the use of synthetic chemical insecticides which not only cause serious harm to environment and human health but also develop resistance against these agents (Aydin and Gurkan, 2006). Indiscriminate use of these chemical has also led to resistance in S. litura population in different geographical areas of the Punjab (Pakistan) (Shad et al. 2012; Ahmad and Mehmood, 2015). This situation demands to evaluate safer eco-friendly alternatives.

Nucleopolyhedrosis viruses (NPVs) can be potent alternatives to the synthetic insecticides against S. litura (Ahmad et al. 2018). NPVs belong to family baculoviruses (BV), which is specific against a variety of insect pests of forests and economically important crops (Tang et al. 2011). This family comprises 600 viruses, including two genera, NPV and Granuloviruses (GV) (Hu et al. 2003). NPVs have great potentials against many lepidopterous pests (Tang et al. 2011 and Zhang et al. 2015).

Emamectin benzoate is a semi-synthetic derivative of abamectin which has been developed for the purpose of controlling variety of lepidopterous pests worldwide (Jansson et al. 1997). Moreover, new chemical insecticides have been established from natural sources; disturb the normal physiological functions of the targeted species (Thompson et al. 2000). Environmental Protection Agency (EPA) has classified emamectin benzoate as a reduced-risk compound due to environmentally benign characteristics (Saunders and Bret, 1997). It has both stomach and contact mode of actions, primarily acting on nicotinic acetylcholine receptor and subsequently on butyric acid receptors. Emamectin benzoate in contrast to synthetic insecticides, has low mammalian toxicity and exerts no toxic effect on non-target organisms. Due to strong mode of action, it is widely used against lepidopterous and dipterous insect pest (Stanley et al. 2006; Wang et al. 2013). In Pakistan, emamectin benzoate has been registered for the control of several species of Lepidoptera in field crops and vegetables.

Integration of synthetic insecticides with baculovirus enhances the efficacy of baculovirus, especially against S. litura (Arti and Yogita, 2014; Shaurub et al. 2014; Ayyub et al. 2019 Nawaz et al.,2019). Keeping in view the importance of low input based crop production and reduction of pesticides’ load on the vegetable crops, the present study aimed to assess the efficacy of NPV alone and in combination with emamectin benzoate against 2nd and 4th larval instars of S. litura from various localities of Punjab (Pakistan) under laboratory conditions.

Material and methods

Insect culture

Larvae of S. litura were collected from three different localities viz. Faisalabad (FSD), Multan (ML), and Layyah (LY), Pakistan and shifted to the Microbial Control Laboratory, Department of Entomology, University of Agriculture, Faisalabad. S. litura was mass cultured under the laboratory conditions at 26 ± 2 °C, 70 ± 5% relative humidity (R.H.) and a photoperiod of 12:12 (L:D) h on a semi artificial diet, following the method of Saljoqi et al. (2015). Adults of S. litura were fed on 10.0% honey solution and provided with a coarse surface of tissue as nappy liner to lay eggs.

Insecticide used

The commercial liquid formulation of Proclaim® (Emamectin benzoate, 19 g l−1, 19EC; Syngenta Pakistan, Ltd., Karachi, Pakistan) was used in the experiment. The insecticide was applied at 0.1 ppm.

Viral isolation and suspension preparation

NPV isolates were obtained from NPV-infected larvae stored in the Microbial Control Laboratory. The presence of NPV was confirmed by inverted microscope (× 40) with Giemsa staining (Yaman et al. 2001). Virus isolation and propagation were carried out in vivo as described by Monobrullah and Nagata (2000). Purified occlusion bodies (POBs ml−1) were counted five times, using a hemocytometer under inverted microscope. A dilution of various concentrations (2 × 109; 3 × 109; and 4 × 109 POBs ml−1) was prepared in distilled water from stock suspension (Cory and Myers, 2003).

Bioassay

Efficiency of emamectin benzoate and NPV was evaluated against 2nd and 4th larval instars. Both pathogens were applied, using a diet incorporation method. A diet piece of 0.5 g was offered to the larvae placed in glass vials (7 cm height, 3 cm in diameter) (Qayyum et al. 2015). The artificial diet, mixed with the three concentrations of NPV (2 × 109, 3×109, and 4 × 109 POB ml−1) and emamectin benzoate (0.1 ppm) or a combined suspension of these was used. Each treatment contained 15 larvae (as a replicate), and was repeated thrice. Larvae were fed on the treated artificial diets for 48 h. After 48 h, survivors were transferred into new vials containing non-treated fresh diet. Bioassay were conducted at 25 ± 2 °C, 65 ± 5% R.H. and a photoperiod of 14:10 (D:L) h in an incubator. Mortality rate was recorded every 24 h and the last count was recorded till pupation of both larval instars. The larvae were poked with a blunt needle and those unable to move in a coordinated manner were considered as dead (Ma et al. 2008; Qayyum et al. 2015). Pupation and adult emergence data were also recorded hereafter. The emerged adults were allowed to mate freely at each treatment and egg hatching percentage was calculated.

Statistical analysis

Mortality means were corrected using Abbott’s formula (Abbott, 1925) and the data was subjected to one-way analysis of variance (ANOVA), using Minitab software (Minitab, 2002) and significance of means were separated by Tukey’s HSD test at 1.0% significance level (Sokal and Rohlf, 1995).

Results and discussion

Efficacy of NPV and emamectin benzoate was challenged against 2nd and 4th larval instars of S. litura on the artificial diet and all treatments were found significant. Highest mortality was recorded for NPV-3 + EB, followed by NPV-2 + EB, NPV-1 + EB, EB, NPV-3, NPV-2, and NPV-3 at all the tests. For 2nd instar larvae, the highest larval mortality rate (49.52%) was recorded for emamectin benzoate in FSD population (F7,71 = 118.0, p ≤ 0.01), followed by LY (F7,71 = 97.7, p ≤ 0.01) and then ML (F7,71 = 112, p ≤ 0.01) populations (Table 1). In the combined treatments, maximum larval mortality (100.0%) was recorded at the highest concentration of NPV (4 × 109 POB ml−1) and emamectin benzoate (0.1 ppm) (Table 1). In case of 4th instar larvae, significant differences were found among all the treatments. In individual applications, the highest larval mortality (44.42%) was recorded for emamectin benzoate in FSD population (F7,71 = 128.0; p ≤ 0.01), followed by LY (F7,71 = 87.6; p ≤ 0.01) and then ML (F7,71 = 121; p ≤ 0.01). Similarly, the highest mortality (96.19%) was recorded in FSD population, followed by LY and ML populations for a high concentration of NPV (4 × 109 POB ml−1) with emamectin benzoate (0.01 ppm) (Table 1). With regard to proportion of insects succeeded to pupate (Table 2), the interaction of NPV and emamectin benzoate varied greatly and significantly.

Table 1 Mean mortality (%±SE) of second and fourth larval instars of Spodoptera litura from three different field populations treated with nucleopolyhedrosis virus and emamectin benzoate (NPV-1 2 × 109 POBs ml−1, NPV-2 3 × 109 POBs ml−1, NPV-3 3 × 109 POBs ml−1, EM 0.1 ppm)
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Table 2 Mean pupation (%±SE) of second and fourth larval instars of Spodoptera litura from three different field populations treated with nucleopolyhedrosis virus and emamectin benzoate. (NPV-1 2 × 109 POBs ml−1, NPV-2 3 × 109 POBs ml−1, NPV-3 4 × 109 POBs ml−1, EM 0.1 ppm)
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The combined applications of microbial pathogens may enhance their pathogenicity, persistence and infection rate (Ali et al. 2016). Moreover, the issue of insecticide resistance can also be minimized as they exhibit novel modes of actions, which is lacking in conventional chemical insecticides (Qayyum et al. 2015 and Bala et al. 2018).

Combined action of both the two pathogens was proved to be more fatal than their sole application and lower proportion of pupation was observed for all populations (2nd instar: FSD 0.00 ± 0.00, LY 2.96 ± 0.27, ML 7.40 ± 0.73; 4th instar: FSD 3.70 ± 0.37, LY 8.88 ± 0.71, ML 13.33 ± 0.96) than the control treatment (˃ 94.0%). Larval mortality increased as the numbers of polyhedral occlusion bodies (POBs) were increased in individual or combined applications. Similar findings were reported by Nawaz et al. (2019) who reported a high mortality of Helicoverpa armigera (H.) larvae with the increase of POBs number. Obtained results are also parallel to the findings of Arrizubieta et al. (2016), who reported the highest effectiveness of NPV and insecticides mixture against H. armigera. This might be attributed to the fact that the polyhedral bodies of NPV attach to the midget of host and multiply, thereby destroying the gut cells. The midgut is the first binding site of POBs, where they multiply and then infection transfer from cell to cell, causing the death of the host (Arif et al. 2018). In the present study, a high mortality rate was recorded in the combined treatments than in the sole application of NPV or emamectin benzoate. Additive or synergistic effects can be resulted by dual action of both pathogens, which may broaden the action spectrum. Similar findings were also reported (Qayyum et al. 2015; Ayyub et al. 2019 and Nawaz et al. 2019; Maqsood et al. 2019). Contrarily, Trang et al. (2002) reported antagonistic effects of NPV and imidacloprid. Antagonism may be caused due to reducing feeding or changing in pH of gut (El-Helaly and El-bendary 2013).

Apart from untreated larvae, the highest level of pupation (2nd instar: FSD 71.85 ± 2.89, LY 79.25 ± 2.59, ML 85.18 ± 2.42; 4th instar: FSD 78.51 ± 1.85, LY 85.18 ± 2.67, ML 91.11 ± 3.22) was observed in NPV1 treatment for both 2nd and 4th larval instars respectively (Table 2). Adult emergence and egg eclosion of both instars were found inversely related to pathogenicity of NPV and emamectin benzoate. Significant differences were observed at individual and combined treatments for all populations. Lowest adult emergence (2nd instar: FSD 0.00 ± 0.00, LY 1.48 ± 0.17, ML 4.44 ± 0.21; 4th instar: FSD 0.00 ± 0.00, LY 6.66 ± 0.13, ML 9.62 ± 039) and egg hatchability (2nd instar: FSD 0.00 ± 0.00, LY 0.00 ± 0.00, ML 2.93 ± 0.09; 4th instar: FSD 4.85 ± 0.08, LY 10.42 ± 0.47, ML 16.19 ± 0.92) was recorded in larvae treated with NPV-3 and EMB in 2nd and 4th instars, respectively, whereas the highest level was recorded in larvae treated with NPV-1. Overall, combined application exerted more hazardous effect on adult emergence and egg eclosion than the individual applications and was inversely proportional to the pathogenicity dependent manners (Tables 3 and 4). Similar findings were also observed by Qayyum et al. (2015) with Bacillus thuringiensis and NPV against H. armigera. Different susceptibility levels were observed in various populations. This might show the level of resistance in insects towards insecticides to which they have been exposed frequently and successively. Resistant populations (ML), frequently exposed to the insecticides, attained cross resistance against emamectin benzoate and vice versa.

Table 3 Mean adult emergence (%±SE) of second and fourth larval instars of Spodoptera litura from three different field populations treated with nucleopolyhedrosis virus and emamectin benzoate. (NPV-1 2 × 109 POBs ml−1, NPV-2 3 × 109 POBs ml−1, NPV-3 4 × 109 POBs ml−1, EM 0.1 ppm)
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Table 4 Mean egg eclosion (%±SE) of second and fourth larval instars of Spodoptera litura from three different field populations treated with nucleopolyhedrosis virus and emamectin benzoate. (NPV-1 2 × 109 POBs ml−1, NPV-2 3 × 109 POBs ml−1, NPV-3 4 × 109 POBs ml−1, EM 0.1 ppm)
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Conclusion

Integration of NPV and emamectin benzoate could be effectively used against the notorious pest (S. litura). Both of the two pathogens represented better tools to combat resistance related issues. Further evaluations under field conditions is needed for further validation.