Background

The tropical armyworm, Spodoptera litura Fabricius (Noctuidae; Lepidoptera), which is one of the important species of genus Spodoptera attacks a variety of agricultural crops, such as horticultural plants, fiber crops, vegetables, and miscellaneous wild plants as well as weeds (Zhou et al. 2010). Its common hosts are cotton, cabbage, lucerne, chickpea, beet, soybean, tobacco, and okra (Ellis 2005). The larvae of S. litura can cause 26–100% yield loss in field (Tuan et al. 2014). It is distributed worldwide, especially in North America, Oceania islands, Africa, and Asia, (El-Helaly and El-bendary 2013) in the sub-continent (Kranthi et al. 2002).

S. litura is an emerging insect pest of Pakistan as it causes a heavy loss in various regions such as the northern and southern districts of Punjab (Ahmad et al. 2007). In Pakistan, S. litura can be found on the cotton crop at the beginning of the cotton season (Saleem et al. 2016). As a sporadic pest, it can infest the cotton crop at any stage. Its overlapping generations throughout cotton cultivated areas of Pakistan decimated this crop in 2003 (Ahmad et al. 2007). The increase in cultivation of succulent crops like cotton, soybean, cabbage, mung bean, and vegetables provides ideal conditions for S. litura to vigorously reproduce, resulting in a rapid increase of generations and population size (Gao et al. 2004).

Despite advancement in pest management techniques, only a few control strategies have been recommended to manage the S. litura populations (Prayogo et al. 2005). Excessive pesticide use resulted to resistance development and environmental and human health issues (Sabir et al. 2011). Increased pest resistance development and environmental problems open opportunities for bio-pesticides (Jacobson et al. 2009). Microbial insecticides, especially virus-based insecticides, are effective biological agents against various agricultural as well as forest insect pests due to their specificity. These viruses are especially used to control lepidopteran insect pests (Liu et al. 2006) and considered as the most intensively studied insect pathogenic viruses (Inceoglu et al. 2006).

Nuclear polyhedrosis viruses (NPVs) belong to the Baculoviridae family (O’Reilly et al. 1992) and are the pathogens which infect many insect pests and other arthropods. NPVs are rod-shaped double-stranded DNA viruses which infect arthropods (Jehle et al. 2006). Many lepidopteran pests, like S. litura (Fabricius), S. exigua (Hübner), S. littoralis (Boisduval), and Helicoverpa armigera (Hübner) have shown susceptibility to NPV isolates (Kumar et al. 2011; Khattab 2013; Ahmad et al. 2018). NPVs can persist in the environment and formulate and package in a similar way to chemical pesticides.

Propagation of NPVs is primarily done in vivo and in vitro (Ikonomou et al. 2003). The most commonly used method is in vivo, simply infecting the healthy larvae by NPV contaminated artificial diet and harvesting the infected larvae for virus propagation (Eberle et al. 2012). The pathogenicity of V-SpltNPV on cotton leaves against the larvae of S. litura was assessed because there is no record about the use of NPV against S. litura on cotton crop in Pakistan. Only a few studies showed the use of NPV (not on cotton) against pests in Pakistan in which the commercially available products were used. Some commercial products have become available in the market.

This current study aimed to evaluate effective control tools of S. litura and to provide basis for research and development on indigenous NPV-based biopesticide in Pakistan.

Materials and methods

Rearing of Spodoptera litura

Larvae of S. litura, collected from various cotton fields in the district Faisalabad, were brought into the laboratory for rearing under controlled conditions. Larvae were reared on an artificial diet (Saljoqi et al. 2015) under controlled conditions (26 ± 2 °C, 70 ± 5 RH, 12:12 h light:dark photoperiod) in IPM laboratory, Department of Entomology, University of Agriculture Faisalabad, Pakistan. The artificial diet consisted of yeast powder (24 g), kidney bean flour (150 g), methyl-4-hydroxy benzoate (1.5 g), ascorbic acid (2.35 g), sorbic acid (0.75 g), formaldehyde solution, agar (8.4 g), streptomycin (0.75 g), and distilled water 550 ml. Newly hatched larvae were transferred individually to plastic vials (3.2-cm height, 3-cm diameter) containing a piece of artificial diet.

Insect virus

The NPV-infected larvae of S. litura were collected from district Vehari, Punjab, Pakistan. Infected larvae were stored in labeled plastic vials and brought to the laboratory where they were placed in a freezer at − 40 °C. Larvae showed infection symptoms were brought to the laboratory and observed to confirm the presence of NPV by inverted microscope (× 40) with Giemsa staining (Yaman et al. 2001). The collected isolate V-SpltNPV (V, Vehari; SpltNPV, S. litura NPV) was used in the laboratory experiment. The name was given to the isolate with reference to the location from which it was collected (V, Vehari; SpltNPV, S. litura NPV). Thereafter, virus isolation and propagation were carried out in vivo as described by (Monobrullah and Nagata 2000). Purified occlusion bodies (OBs/ml) were counted 5 times using a hemocytometer under inverted microscope. A dilution of various concentrations (1 × 104 to 1 × 108 OBs/ml) of V-SpltNPV was prepared in distilled water from stock suspension (Cory and Myers 2003).

Laboratory bioassay

S. litura larvae were obtained from the laboratory rearing colony. Cotton leaves 3 cm in diameter were cut and placed in a plastic container (7-cm height and 3 cm in diameter). Various concentrations (1 × 104, 1 × 105, 1 × 106, 1 × 107, and 1 × 108 OBs/ml) were prepared and 5–10 μl viral concentration was applied on leaf disks with a micropipette. Control treatment were applied, using only distilled water. Newly molted 30 larvae of 2nd, 3rd, and 4th instars (30 for each instar) were placed in a container having treated leaf disk and allowed to feed on contaminated leaf disks. After 24 h, larvae were shifted on fresh leaves. Fresh leaves were provided daily until pupation. All plastic containers were placed in a growth chamber under controlled conditions (25 ± 2 °C, 70 ± 5% R. H, and 14:10 (D:L) photoperiod). LC50 and LT50 values were calculated from mortality data after every 48 h. All experiments were replicated 3 times.

Greenhouse evaluation of NPV alone and in combination with spinosad

The bioassay was performed under greenhouse conditions, using potted cotton plants of the same age (50 days). Three concentrations (1 × 106, 1 × 107, and 1 × 108 OBs/ml) of V-SpltNPV were used for greenhouse experiments. For evaluation of the effectiveness of spinosad, (Tracer 240 SC, Dow AgroSciences) it was used alone and in binary combination at the 3 different concentrations of V-SpltNPV (1 × 106, 1 × 107, and 1 × 108 OBs/ml). The 20 ml water was calibrated for spraying the whole cotton plant. For the greenhouse experiment, 1 ml of each virus concentration was mixed with 19 ml distilled water to make 20 ml final volume. For treatment applications of spinosad (recommended dose; 1%), 20 ml formulation was prepared and sprayed on potted cotton plants. For the combined application of spinosad with different concentrations (1 × 106, 1 × 107, and 1 × 108 OBs/ml) of NPV (the recommended dose of spinosad was mixed with different concentrations of NPV) comprised of a 20-ml solution, applied along with 0.1% Tween-80 as adjuvant. Twenty larvae of 2nd instar S. litura were released separately on each potted cotton plant with a camel hair brush. Cotton plants were covered by a 0.5 mm2 meshed mosquito net to avoid larval escape. The concentrations were sprayed on cotton plants by a hand sprayer. Mortality rate was observed daily until pupation.

Statistical analysis

Corrected mortality rates were calculated by Abbott’s formula (1925) and data were analyzed using Minitab software for LT50 and LC50 values. For laboratory and greenhouse trials, data were analyzed, using Statistica 8.1 software and means were separated by Tukey’s HSD test at α = 5%.

Results and discussion

LC50 values of different larval instars of S. litura, when exposed to various concentrations of V-SpltNPV, are presented in Table 1. Larval mortality was high in early instars and decreased in full-grown tested larvae. Maximum mortality rate (88.08%) for 2nd instar larvae was observed, while the minimum mortality rate (65.52%) was recorded for 4th instar larvae of S. litura, when exposed to various concentrations. The calculated LC50 were 1.3 × 102, 5.4 × 104, and 6.9 × 105 for 2nd, 3rd and 4th larval instar, respectively. The increase in the LC50 values, with an increase in the age of larvae, may be due to the dilution effect of virus inoculum associated with the increase in weight of larvae. LC50 values also showed that 2nd instar larvae was (53,865 and 689,865) times more susceptible than 3rd and 4th larval instars, respectively, while 3rd instar larvae was (636,000) times suceptible than 4th instar larvae.

Table 1 LC50 values of V-SpltNPV for different instar larvae of Spodoptera litura
Full size table

LT50 for various larval instars of S. litura, when exposed to various V-SpltNPV concentrations, are presented in Table 2. Data in the table showed the concentration and larval instar dependent on LT50 values. The decreasing trend of LT50 values was observed by increase in concentration used in the experiment. Likewise, with the increase in larval age, more time was required to kill the tested population. LT50 of 2nd instar larvae were 4.99 days against the highest concentration (1 × 108 OBs/ml), which increased up to 11.13 days against the lowest concentration (1 × 104 OBs/ml). LT50 values of 3rd instar larvae were 6.61 and 12.35 days and were observed against maximum and minimum concentrations, respectively. The same trend was observed for 4th instar larvae, where LT50 value was 7.49 days against (1 × 108 OBs/ml) concentration, while the LT50 value was increased to 11.11 days against 1 × 104 OBs/ml concentration.

Table 2 LT50 values of Spodoptera litura larvae exposed to various concentrations of V-SpltNPV
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It was evident from the results that the combined application gave better results than each application of tested concentration alone (Fig. 1). The sole V-SpltNPV concentration (1 × 108 OBs/ml) caused 52.63% mean mortality of tested larvae. Similarly, sole application of spinosad caused 46.58% mean mortality. V-SpltNPV (1 × 108 OBs/ml) and spinosad combination caused a maximum mortality of 100%. The combination of a low concentration V-SpltNPV with spinosad also gave effective results and caused 75–78% (1 × 106 OBs/ml) and 91.31% (1 × 107 OBs/ml) mortality rates of S. litura larvae.

Fig. 1
figure 1

Percentage mean mortality of 2nd instar larvae of Spodoptera litura after every 24 h against various concentrations sprayed on cotton plants in greenhouse

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The V-SpltNPV isolate was effective for managing the larval population of S. litura. The LT50 and LC50 were gradually enhanced by increase in larval instar. The increase in the LC50 values with an increase in the age of larvae was due to the dilution effect of virus inoculum associated with an increase in weight of larvae. Monobrullah and Nagata (2000) also noticed an increasing trend of LD50 values for second (224 PIBs/larva) to 5th (519,381 PIBs/larva) instar larvae of S. litura. They also noticed an increase of LT50 for 2nd instar larvae (6.9 days) to 5th instar larvae (9.3 days), when exposed to the same concentration. Similarly, Trang and Chaudhari (2002) reported increasing trends of LC50 (1 × 103 to 1.5 × 109 PIB/ml) and LT50 (4.4–9.4 days) values with the increase in larval instars. The variation of LC50 values may be due to the change in diet as they used coaster leaves in the experiment instead of cotton. In addition, the larvae were exposed at different time intervals to calculate LC50, while in the present study, the larvae were exposed only once. The LT50 values were inversely proportional to dose, while directly proportional to larval instar (Subramanian et al. 2005; Kouassi et al. 2009; Bhutia et al. 2012). A similar trend was observed in other studies as well (Cherry et al. 1997). The LT50 values may deviate to present findings due to change of Spodoptera species which were exposed to NPV.

Mature larvae were more resistant to the concentration of V-SpltNPV due to physiological changes (body mass) related to pupation. Kumar et al. (2011) noticed increasing trends in the LC50 and LT50 values of SpltNPV for 2nd to 3rd larval instars of S. litura. Similar findings were documented by Teakle et al. (1986) who noted the increasing in LC50 values of NPV commercial formulation with an increase in larval instar of Heliothis punctigera (Wallengren). Ahmad et al. (2018) also recorded the increase in LC50 values (2.64 × 103 to 2.15 × 106 OB/ml) and LT50 values (72.50–144.64 h) for 2nd and 5th larval instars of S. litura against various concentrations of SpltNPV. They also stated that the same trend of LT50 value as (72.50 h) for 2nd instar larvae, which increased up to (144.64 h) for 5th instar larvae. Similarly, Evans (1981) reported variation in LC50 values among 1st and 5th larval instars of S. litura. LC50 value was (34,000 times) higher for 5th larval instar, than the 1st instar larvae of Mamestra brassicae (Linnaeus), while 5th and 6th instars larvae were almost at par for resistant to virus infectivity.

The physiological changes (increase in body mass) associated with pupation might not allow infection at the late developmental stage as the mature larvae (10 days) were found to be more resistant to SlNPV (Kumar et al. 2011). The possibility of biovirus not getting sufficient time to replicate or kill the larvae may not be ruled out. Such suggestion gets support from the findings of Evans (1981) and Teakle et al. (1986). In addition, Tuan et al. (1998) and Jayanthi (1992) also observed significant differences in LC50 values among different larval instars of S. litura and also in Trichoplusia ni (Milks et al. 1998).

The combination of spinosad with EPV proved to be suitable because spinosad has no antiviral, antifungal, or antibacterial activity (Bret et al. 1997). Spinosad has been distinguished as a biopesticide, as spinosyns, which are produced by fermentation of soil bacterium actinomycete (Copping and Menn 2000). Spinosad has insecticidal properties that differentiate it from other entomopathogenic bio-pesticides (Salgado et al. 1998).

Bret et al. (1997) noted that the combination of spinosad with SlNPV resulted in better control of S. furgiperda population. The present findings are in agreement with El-Helaly and El-bendary (2013) that combined treatment of SlNPV and spinosad exhibited a maximum larval mortality (55%) of S. littoralis. While alone treatment with SlNPV caused 20.11% mortality and spinosad gave 26.66% mortality of S. littoralis. These findings showed an additive correlation of spinosad with NPV. The combined application of AgMNPV and spinosad also increased the mortality of pickleworms larvae up to 78% (Jackson et al. 2014). While significantly lower mortality rate 32 and 24% was observed against AgMNPV and Spinosad, respectively, when each was applied alone. The additive effects of spinosad and SfMNPV combination was also reported in previous findings of Mendez et al. (2002) against larvae of S. furgiperda as combined treatment caused a high mortality rate than the alone application. Similarly, 40% more control of S. litura was reported, when exposed to the combined formulation of SpltNPV and azadirachtin (Cook et al. 1996). In addition, Nathan and Kalaivani (2005) observed 92.7% mortality rate, when a combined application of azadirachtin (AZA) and NPV was used against S. litura larvae than individual application of NPV (28.5%) and AZA (36.3%). Similarly, Shaurub et al. (2014) suggested that the mixture application of NPV with AZA enhanced larval mortality of S. littoralis significantly as compared to individual treatment.

Conclusions

It is the time to use baculoviruses to manage insect pests in agricultural fields of Pakistan, to reduce the use of synthetic toxic chemicals, especially in cotton-growing regions, where more than 80% of total imported pesticides are being used to manage pests. The isolated strain of NPV (V-SpltNPV) can be effectively used to manage S. litura population. Furthermore, the combined efficacy can be enhanced by evaluating their pathogenicity with new chemistry insecticides without causing damage to non-target organisms.