American bollworm (ABW), Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae), has been observed most dangerous pest and yield decliner of the field crops and vegetables in Pakistan since the 1990s and early 21st century. It has become the most hazardous pest due to its polyphagous nature, migratory behavior, high fecundity rate, multiple generations and can develop the resistance against insecticides. It is estimated that annually, 2.5 million bales losses were caused by this insect pest to the cotton crop (Karim, 2000; Muhammad et al., 2009).

Different levels of resistance (moderate to high) to a wide range of insecticides have been reported worldwide, including Pakistan, among the field population of ABW (Sayyed and Wright, 2006; Damalas and Eleftherohorinos, 2011; Ishtiaq and Saleem, 2011). Therefore, the management of this pest for an extended period with reduced risk of insecticides is the dire need of time and a pragmatically suggested option. Among the various biological control agents used as an alternative to synthetic insecticides (Ali et al., 2015, 2018a,b,c,d,e), the preference for entomopathogenic Nuclear Polyhedrosis Virus (NPV) has been increased in the recent years. Furthermore, new chemistry insecticides have been established from natural sources, disturb the normal physiological functions of the targeted species in pest management programs (Qasim et al., 2018a; Schneider et al., 2003; Thompson et al., 2000; Dhadialla et al., 1998) by targeting insect development mechanism like moulting as well as chloride channel activator. Another advantage of new chemistry insecticide over the broad-spectrum insecticides is that they are selective to target pest species and less dangerous to natural enemies (Grafton-Cardwell et al., 2005).

Among microbial insecticides, the insect pathogenic viruses such as baculoviruses were used successfully for biological control under integrated pest management (IPM) program. It has been used for more than 20 years with great success (Zhang, 1989) in different parts of the world for managing different insect pest species based on their several advantages. These microbial agents are highly host-specific and are known to be entirely safe for humans, animals and non-target beneficial organisms such as bees, predatory insects and parasitoids (Monobrullah and Nagata, 1999; Nakai et al., 2003; Islam et al., 2018a, b). Keeping in view the facts as mentioned above, the present study was carried out to evaluate the effectiveness of NPV against ABW under controlled conditions.

The study was conducted in Integrated Pest Management (IPM) Lab, Department of Entomology, University of Agriculture, Faisalabad, Pakistan during 2016–2018.

ANOVA parameters indicated that all NPV concentrations were significantly effective against the second and third instar larvae of ABW (F=592, P=0.000). Results showed that higher concentrations had given the highest mortality when treated with NPV alone in diet mixed method. It was also observed that the effectiveness of NPV increased with time. Mean percent mortality comparison showed that maximum mortality (95.00±2.05%) was seen at 1×1010POB which was 9.95 times higher than control, followed by 95.00±1.99% at 1×109POB which was 9.95 times higher than control. However, most diluted NPV dose (1×105POB) caused enough larval mortality, reaching up to 78.43±1.20% which was 7.80 times higher than control (10.05±0.39%) (Table 2).

Overall, analysed data indicated that all spinosad concentrations were significantly lethal to the second and third instar larvae of ABW (F=27.4, P=0.000). Mean percent mortality comparison showed that maximum mortality (50.87±0.81%) was observed at (0.8%) which was 13.71 times higher than control followed by 42.10±0.66% at (0.4%) which was 11.34 times higher than control. Though, most diluted dose (0.025%) caused only 22.80±0.37% mortality of ABW larvae, which was 6.14 times higher than control (3.71±0.28%) (Table 1).

ANOVA parameters indicated that the combined effect of all NPV concentrations and spinosad proved to be significant against the second or third instar larvae of ABW (F=680, P=0.000). Mean percent mortality comparison showed that maximum mortality (95.00±2.06%) was observed at NPV6 + Spinosad (1×1010 POB+0.1%) which was 9.91 times higher than control followed by (95.00±1.98%) at NPV5+Spinosad (1×109 POB+0.1%) which was 9.91 times higher than control. Even though the combination of NPV1+Spinosad (1×105 POB+0.1%) was also too much lethal to ABW larvae, causing 80.71±1.91% mortality, which was 7.99 times higher than control (10.09±0.31%) (Table 2).

While using the bio-agent in IPM, there are some limitations such as these agents are slow in action, low persistence in environment and need of a repeated application to infect the host population. The combined use of microbial agents may help in resolving these problems and increasing the pathogenicity, persistence and rate of infection (Ali et al., 2015, 2016). The issue of insecticide resistance can also be minimized by using these agents as they used different tactics to kill or control host, as compared to other conventional insecticides (Bala et al., 2018). ABW is the pest of great importance that attacks many valuable crops.

The resistance of ABW against many insecticides has been noted, and it was the issue in focus for many years. Entomopathogens can control the pest having resistance against insecticides (Cloyd, 2014). The rotation of crops and pesticides can also be helpful to minimize the problem of resistance (Zahn and Morse, 2013). When two or more bio-agents are used in combination, they increase the effectiveness, and host spectrum of each other and reduce the mortality time (Kalantari et al., 2014). It can be imagined that the mutual action of microbes may increase the virulence as expected alone. The polyhedral bodies of NPV attach to the midgut 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 (Liu et al., 2006; Arif et al., 2018; Qasim et al., 2018b; Shakeel et al., 2018).

Among these concentrations, after the exposure to treated diet, mortality was recorded. High concentrations of both NPV and Spinosad gives the highest mortality. Such as NPV gave highest mortality (95%, 95% and 92.15%) at 1×1010, 1×109 and 1×108 POBs concentrations and these results were parallel to those results of Arrizubieta et al. (2016), who reported that a mixture of NPV and insecticides against H. armigera was effective under laboratory condition. These results showed that effectiveness and tenacity of mixture caused significant mortality of larvae. Spinosad gave the highest mortality (95%, 95% and 95%) and 0.8%, 0.4% and 0.2% concentrations and these results were supported by Jat and Ameta (2013). The study results concluded that Flubendiamide 480SC and Spinosad 45SC (at 200mL·ha‾¹) were found to be effective against the larvae of ABW, and these insecticides caused 89.94% and 74.67% mortality, respectively.

The viruses multiply within the body of the host and destroy the host cell tissues. The pathogenicity and rate of response depend upon the dose, temperature and larval body size, and it varies from 4 to 14 days (Jones et al., 1994). Thus the pathogenicity varies with treatment and time, as the dose increase the control efficacy also increases.

Combination of two or more bio-agent can be an option to delay the resistance and increase the effectiveness (Kalantari et al., 2014). In the current study, both additive and antagonistic interactions were observed, and these results were supported with the findings of Wang et al. (2009) who found that lethal and sub-lethal doses of spinosad increase the mortality and delay the developmental process. The additive impact was supported by the results of Wakil et al. (2012), who found that there was an additive effect between NPV and Azadiractum against ABW. Moreover, Qayyum et al. (2015) also reported the additive impact of NPV and Bacillus thuringiensis against ABW. In our study, the antagonistic effect was supported by the outcomes of Trang et al. (2002) as they observed the antagonistic effect of NPV and Imidacloprid. The antagonistic effect might be due to the decrease in feeding potential or pH change of gut (El-Helaly and El-bendary, 2013).

We speculated that microbial control through NPV and Spinosad is an effective approach against ABW. The repeated use of synthetic pesticides against major pests could be the main reason of pest resurgence, and this study results would provide useful insight to build a framework for future investigations and to reduce the use of toxic chemicals for the control of insect pests of major crops.

All authors declare no conflict of interests.