Individual and combined impact of nuclear polyhedrosis virus and spinosad to control the tropical armyworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), in cotton in Pakistan

The tropical armyworm, Spodoptera litura Fabricius (Noctuidae; Lepidoptera), is among the most harmful pests causing economic loss in the quality and production of a variety of crops, particularly cotton. Entomopathogens play an important role in insect pest management. The nuclear polyhedrosis virus (NPV) isolate of S. litura (V-SpltNPV) was isolated from infected larvae in a cotton crop, and viral occlusion bodies were confirmed, using an inverted microscope. The pathogenicity of V-SpltNPV against 2nd, 3rd, and 4th larval instars of S. litura was evaluated at various concentrations (1 × 10⁴ to 1 × 10⁸ OBs/ml). Mortality rate was high (37.65–96.82%) in early instar larvae against tested concentrations. LC₅₀ and LT₅₀ values increased with increasing larval age. There was 689,865 times increase in LC₅₀ value (1.35 × 10² OBs/ml) for 2nd instar larvae to LC₅₀ value (6.90 × 10⁵ OBs/ml) for 4th instar larvae. LT₅₀ values enhanced from 4.99 days for 2nd instar larvae to 7.49 days for 4th instar larvae, due to a decrease in efficacy of NPVs with the increasing age of larvae. In a greenhouse experiment, a combined application of spinosad with V-SpltNPV (1 × 10⁸ OBs/ml) caused (100%) mortality of 2nd instar larvae. A single application of V-SpltNPV (1 × 10⁸ OBs/ml) resulted to mean mortality (52.63%) of tested larvae. The native isolate V-SpltNPV seems to have a potential to be used in integrated manner with other IPM tactics to significantly reduce the use of toxic chemical pesticides.

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.

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%.

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.

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.

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.

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

Full size image

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.

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.

The data used and analyzed during this project are available from the corresponding author on reasonable request.

IPM:

Integrated pest management

Kbp:

Kilobase pare

LC50:

Lethal concentration to kill 50% population

LD50:

Lethal dose to kill 50% population

LT50:

Lethal time to kill 50% population

NPV:

Nuclear polyhedrosis virus

OBs/ml:

Occlusion bodies/milliliter

PIB/ml:

Polyhedral inclusion bodies/milliliter

S.E:

Standard error

V-SpltNPV:

Vehari-Spodoptera litura nuclear polyhedrosis virus

χ2:

Chi square

The authors highly acknowledged Dr. Laura L Ingwell from Purdue University, Indiana, USA, for review and her valuable comments for this article.

Funding

Funding Agency: Higher Education Commission of Pakistan

Funding Number: No.20-3209/NRPU/R&D/HEC/13/460

Affiliations

1. Integrated pest management Laboratory, Department of Entomology, University of Agriculture Faisalabad, Faisalabad, Pakistan
   • Muhammad Bilal Ayyub
   • , Ahmad Nawaz
   •  & Muhammad Jalal Arif
2. Virology Laboratory, Department of Plant Pathology, University of Agriculture Faisalabad, Faisalabad, Pakistan
   • Luqman Amrao

Contributions

MBA and AN designed the experiment. MBA conducted the experiment and wrote the article. AN helped in statistical analysis. MJA and LA revised the article. All authors approved the final article after reading.

Corresponding author

Correspondence to Muhammad Bilal Ayyub.

Ethics approval and consent to participate

N/A

Consent for publication

N/A

Competing interests

The authors declare that they have no competing interests.

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