Molecular Identification and Pathological Characteristics of NPV Isolated from Spodoptera litura (Fabricius) in Pakistan

Jam Nazeer Ahmad1,2,*, Rashid Mushtaq1, Samina Jam Nazeer Ahmad1,2, Sumaira Maqsood3, Ishita Ahuja4 and Atle M. Bones4

1Intergrated Genomics Cellular Developmental and Biotechnology Lab, Department of Entomology, University of Agriculture, Faisalabad

2Plant Stress Physiology and Molecular Biology Lab, Department of Botany, University of Agriculture, Faisalabad

3Institute of Agricultural Sciences, University of the Punjab, Lahore

4Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim, Norway

ABSTRACT

The cotton army worm Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is a destructive pest of various field crops and vegetables in Pakistan. Development of biopesticide is an attractive strategy to minimize the problems of pest resistance, environmental pollution and human health concerns. The isolates of S. litura nucleopolyhedroviruses (SltNPV) were collected from infected larvae fed on natural cotton crops. The NPV was isolated from the larvae and viral occlusion bodies (VOBs) were detected using a light microscope. The toxicity of native isolates against S. litura also studied by testing different concentrations (1 x 102 POB (Polyhedral occlusion bodies) mL-1- 1 x 1010 POB mL-1) from the occlusion bodies produced from NPV isolates against 2nd, 3rd, 4th and 5th instar larvae of S. litura. The rapid and sensitive polymerase chain reaction (PCR) technique was used for the molecular detection of NPV gene from native NPV diseased insect. Multiple sequence alignment and phylogenetic analysis were performed to compare SlNPV- FSD15 based on Lef-8 with other Lef-8 genes sequences clearly showed that our SlNPV-FSD15 isolate belongs to Spodoptera litura associated NPVs. The biological activities of this NPV isolates were investigated under laboratory condition. The highest mortality of S. litura was observed at early instars. Against second instars of S. litura, LC50 values of NPV isolate ranged from 1.92×103 to 3.64×103OB/ml with LT50 values of 69.30 h to 72.80 h, respectively. This study showed highly effectiveness and provides an opportunity to cut down the use of synthetic approaches and develop safe biological/microbial insecticides from NPV isolates, which in future may effectively control S. litura.

Article Information

Received 28 May 2018

Revised 21 June 2018

Accepted 10 July 2018

Available online 05 October 2018

Authors’ Contribution

JNA, RM and SJNA presented the ideas and wrote the manuscript. RM, SJNA and SM reviewed the literature. JNA, IA, and AMB critically analyzed and edited the manuscript.

Key words

Spodoptera litura, PCR, NPV, Biopesticide, DNA barcoding, Nucleopolyhedro-viruses.

DOI: http://dx.doi.org/10.17582/journal.pjz/2018.50.6.2229.2237

* Corresponding author: jam.ahmad@uaf.edu.pk

0030-9923/2018/0006-2229 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan

Introduction

Spodoptera litura Fabricius (Lepidoptera: Noctuidae) is also known as pigweed caterpillar or tobacco cutworm is cosmopolitan and polyphagous pest which cause severe losses in southern and northern districts of Pakistan (Ahmed et al., 2016; Saleem et al., 2016) and it is widely distributed throughout Asia, Africa and Europe (Nathan et al., 2005; El-Helaly, 2013). It is a destructive pest of numerous economically important cash crops such as cotton, groundnut, soybean, tomato, sweet potato, onion, clover chili, cauliflower and cabbage (El-bendary and El- Helaly, 2013; Saleem et al., 2016). In vegetables, it attacks leaves, stem, fruiting points and occasionally, also seed pods resulting in considerable yield loss. Current methodsto control this pest are based on the use of conventional insecticides which causes the development of resistance in the pest and further has a negative environmental and human impact. Extensive use of synthetic insecticides has led to outbreaks of insecticide resistance in S. litura (Bhatti et al., 2013; Ahmed et al., 2015). The development of resistance in S. litura against organochlorine, organophosphates and synthetic pyrethroids has been reported in the areas of cotton belt of south Punjab, Pakistan (Ahmed et al., 2015). Thus this situation demands to search out some ecological safe alternative like microbial pathogen to control this notorious pest. An attractive and alternate tool for S. litura control is the use of entomopathogens. Several species of insect viruses, bacteria, fungi and protozoa were isolated and investigated for control of S. litura (Çakici et al., 2014). In recent years, more than 600 insect species belonging to order Lepidoptera, Hymenoptera, Diptera, Orthoptera, Coleoptera, Neuroptera, Thysanura, and Trichoptera infected with baculovirus have been isolated (Haase et al., 2015). Baculoviruses are insect-specific, diverse group of double-stranded DNA circular viruses with genome in the region of 80-180 kbp. Baculoviruses belong to the family baculoviridae which is further divided into two genera, the Nucleopolyhedroviruses (NPVs) and Granuloviruses (GVs) (Hu et al., 2003; Jehle et al., 2006; Barreto et al., 2005). The NPV s are cuboidal shaped have occlusion body of 0.4 to 2.5 µm in size visible under electron microscope (Moser et al., 2001; Shapiro et al., 2004). Lepidopteran pests, Spodoptera exigua (Murillo et al., 2001; Wu et al., 2012; Khattab, 2013). S. litura (Lavina et al., 2001; Martins et al., 2005; Laarif et al., 2011), Helicoverpa armigera (Lepidoptera: Noctuidae) (Mehrvar et al., 2007; Kumar et al., 2012; Noune and Hauxwell, 2015) and Malacosoma americanum (Demir et al., 2014) have shown susceptibility to several isolates of NPV. Horizontal transmission of baculoviruses from one host to another occurs when a susceptible insect ingests food contaminated with occlusion bodies (OBs) (Takahashi et al., 2015). Several studies have reported the isolation of NPVs from S. litura (Lavina et al., 2001; Martins et al., 2005; Lucien et al., 2009; Laarif et al., 2011; Kumar et al., 2011), H. armigera (Figueiredo et al., 1999; Ogembo et al., 2007; Mehrvar et al., 2008; Kumar et al., 2012; Noune and Hauxwell, 2015) S. exigua (Murillo et al., 2001; Wu et al., 2012; Khattab, 2013), Trichoplusia ni (Erlandson et al., 2007) Diaphania pulverulentalis (Pachippan et al., 2012), Turnip moth (Jakubowska et al., 2005) and Chrysodeixis includens (Alexandre et al., 2010).

All NPVs have molecularly been characterized by restriction endonuclease mapping of viral DNA, showing that they can be distinguished from each other by one or more DNA restriction enzyme fragments. At biological level, some of these strains have presented better insecticidal activities, which make them more suitable to S. litura (Martins et al., 2005; Laarif et al., 2011) H. armigera (Ogembo et al., 2007; Mehrvar et al., 2008) and S. exigua control (Khattab, 2013). The present study was planned to identify NPVs as a biological control agent of S. litura from local Spodoptera NPV infected population. Therefore, a simple molecular procedure suitable for diagnosis of viruses in their natural hosts was adopted and the evaluation of its biological activity against different instars is being reported.

 

Materials and methods

NPV diseased insect collection

The original virus isolates were obtained from diseased S. litura larvae collected from cotton field of PARS-UAF. The larvae that showed baculovirus infection symptoms were brought to laboratory (Fig. 1A) and examined to confirm the presence of virus by light microscope with Giemsa staining according to Mustafa et al. (2001), in which a thin smear of infected worm tissue was mounted and air dried. The smear was immersed for 1-2 min in Giemsa, rinsed under running tap water for 5-10 sec then the smear was stained for two hours in 10% Giemsa stain (10g of Giemsa dissolved in 100ml distilled water). The dye was rinsed off in running tap water for 5-10 sec and allowed to air dry, then examined under a light microscope to detect the Occlusion Bodies (OBs). After the examination, the diseased larvae were kept at -20°C until the purification of OBs (polyhedra).

Light microscope examination

Moribund larvae showing specific disease symptoms were individually examined for the presence of polyhedral inclusion bodies using light microscope. A wet smear of the homogenized liquid using a drop of haemolymph or a small part of larval tissue was spread on a glass slide. The slide was then dipped in 10% Giemsa’s stain for 10 min. The excess stain was then washed with running water for 5-10 sec (Wigley, 1976). The prepared smear was examined using the oil immersion of phase contrast microscope. The smear test would allow recognition of the occlusion bodies of nuclear polyhedrosis viruses NPV.

NPV production and purification

The diseased larvae of 3rd to 5th instar of S. litura were collected from cotton fields in Faisalabad, Pakistan, in 2013. The infected larvae showing baculovirus presence symptoms were brought to IGCDB laboratory and stored at -20 °C until the isolation of OBs (polyhedra). Isolation and purification of the NPV was performed in 4th instar of S. litura. Using haemocytometer (Hausser Scientific), NPV viral concentrations were quantified under a light microscope with six counts per hemocytometer. A stock suspension of NPV was prepared. A dilution of various concentrations 1 x 102- 1 x 109 OBs/ml concentration was prepared from the stock suspension. Twenty-five larvae were placed in 50 ml tube and homogenized. But, for NPV isolation from S. litura infected samples, larvae were treated with 0.1% SDS (1 ml/larva) for 1 night at 4°C and filtered through 5 layers of cheese cloth. POBs were settled at bottom of tube by centrifugation at 3600g for 10 min at room temperature in 50 ml centrifuge tubes. Before final isolation, the pellets were re-suspended in 0.5% SDS and centrifugation and re-suspension were repeated with 0.3 M NaCl. Then the OBs were finally re-suspended in distilled water. Then, following the protocol of Cheng et al. (1990), POBs were further purified according to sucrose gradient centrifugation method.

 

DNA extraction and PCR

DNA was extracted from NPV infected larvae of S. litura with DNeasy Tissue kit (Qiagen, Hilden, Germany) according to manufacturer protocols. Quantity and quality of extracted virus DNA was estimated using UV spectrophotometer (Pico200) and by the ethidium bromide 1.5% agarose gel electrophoresis, respectively. The DNAs obtained were stored at -20°C until used. The primer nucleotide sequences based on NPV conserved gene late expression factor (Lef-8) was carried out for PCR in a reaction volume of 48 µl. The reaction volume contains ddH2O (33 µl), 10Xreaction buffer (5 µl), 1 mM dNTPs (5 µl), 200 µM of each forward and reverse primers (1 µl), and 1 µl of purified DNA (~ 50 ng). The lef-8 specific degenerate primers (prL8-1 and prL8-2) developed by Lange et al. (2004) was used in PCR. The amplification of DNA was accomplished with a PCR thermal cycler (PeqStar, Germany) under following PCR cycling conditions. DNA samples were preheated to 95°C for 4 min, followed by 35 cycles with denaturation temperature of 95°C for 2 min, annealing temperature of 46°C for 2 min, and extension temperature at 72°C for 1 min. Then, final extension at 72°C for 2 min was included for final amplification. In the first step of PCR, 0.2 µl of Taq polymerase and 1.8µl its dilution buffer was added in each tube making final reaction volume up to 50 µl. The PCR product was analyzed by 1% agarose gel electrophoresis at 80 V for 1 h. After the PCR, the gel was stained with ethidium bromide and photographed under a UV light. Gels were stained in solution of ethidium bromide (2µg/mL) and were visualized under UV light using fisher scientific gel documentation system (Syngene™ IG3) for the confirmation of virus presence.

Nucleotide sequencing and phylogenetic analysis

PCR product (745 bp) was purified using commercial kit and sequencing was performed and analyzed on AbiPrism 3100 Genetic Analyzer apparatus (Applied Biosystems, USA). The resulting Lef-8 sequence has been deposited in international database (NCBI). NPV Lef-8 sequences identified by Blast sequence homology were downloaded from Genbank database (http://www.ncbi.nlm.nih.gov). All Lef-8 sequences were aligned with ClustalW and phylogenetic analysis was performed under MEGA6 software (Tamura et al., 2013).

Insect rearing and bioassay study

The S. litura larvae used in this experiment derived from cotton fields of Punjab and reared on artificial diet consisting of chickpea flour 150g, sorbic acid 0.75 g, yeast powder 24g, linseed oil 6 ml, agar 8.4 g, vitamin mixture 0.02 g, ascorbic acid 2.35 g, methl-4-hydroxy benzoate 1.5 g, d H2O 550 ml and streptomycin 0.75 g. The rearing was done under controlled laboratory condition (25± 2°C, 70± 5 RH, 14:10 h light: dark photoperiod). The diet was stored at 4°C until use. About fifty 2nd and 3rd instar S. litura larvae were transferred in individual plastic vials containing 0.5-1.5 g thin layer of artificial diet until they reached at fourth larval stage. Then, they were transferred to boxes containing 3 cm thick layer of vermiculite (<0.5 mm grain size) for pupation. Additional artificial diet was provided shortly before pupation. Pupae were collected and incubated at 25°C. All the larvae were maintained at 25±2°C, 75% r.h and 16:10 (D:L) photoperiod (Jehle et al., 2013).

All the bioassays were performed according to Lucien et al. (2009) with slight modification. Briefly, Virulence of NPV was tested against 2nd, 3rd 4th and 5th instar larvae of S. litura. Molted larvae were distinguished by their head capsule slippage and transferred to individual vials for 12 h. Different NPV suspensions (1 x 102; to 1x 109 POB mL-1) were prepared and 5-10 µl of each viral concentration was socked with fresh piece of artificial (2mm2) in vials. Molted Larvae were allowed to feed on the treated artificial diets until complete consumption. After consuming artificial diet, 25 larvae were individually shifted on diet without virus suspension. Larvae served with virus free diet constituted the healthy controls. LC50 and LT50 values were determined from data observed every day. Concentrations of OBs were determined with haemoctometer under phase contrast microscopy at 400X magnification. All the treatments were replicated thrice and maintained at 25±2°C, 75% r.h. and L16: D8 h photoperiod.

Statistical analysis

The data were analyzed using Probit analysis software to arrive at lethal concentration of virus required to cause 50% mortality (LC50) and lethal time required to cause 50% mortality (LT50)

 

Results

NPV isolates and VOBs

The native S. litura NPV isolates were obtained from infected larvae collected from cotton field of Post graduate Agriculture Research Station (PRAS) of University of Agriculture Faisalabad. The symptoms of viral infection on S. litura collected from the cotton field were liquefied ruptured larval body (Fig. 1A), and slow motion swollen larvae with red color cuticle (Fig. 1B). The field collected virus isolate was cultured in a S. litura laboratory colony (Fig. 1C-F). Then infected propagated colony was then purified and kept at -80°C for further studies.

The viral occlusion bodies (VOBs) from diseased larvae were observed under a light as well as inverted microscope. VOBs appeared as polyhedral and negatively stained particles after Giemsa staining. Under light and inverted microscope, NPV associated occlusion bodies were observed (Fig. 2).

 

Amplification of NPV specific gene

The infected samples collected from Faisalabad district were analyzed for PCR detection. The partial codons sequence of the late expression factor-8 (Lef-8) gene was amplified using specific PCR primers as a single fragment at correct length (~745 bp) (Fig. 3).

DNA sequencing and phylogenetic analysis

The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). Phylogenetic analyses were conducted in MEGA6 (Tamura et al., 2013) for the comparison of the SlNPV-FSD15-lef-8 gene sequences with the sequences of other lef-8 genes of various nucleopolyhydroviruses (NPVs) available in GenBank. The nucleotide sequence of the native NPV isolate (Slt NPV-FSD15) is composed of 745 bp, submitted at Gen Bank NCBI. This sequence was aligned using ClustalW with the NCBI available nucleotide sequences of NPVs associated with different insects. The molecular phylogeny of nucleotide sequence of the SlNPV-FSD15-lef-8 showed maximum homology (99-100%) with nucleotide sequence of SpliNPV from S. litura of Germany, Japan and India origin (AY706581.1, AB326103.1, JF2760358.1, AB583682.1, AB581187.1). The Helicoverpa armigera associated NPV formed separate clade clearly distinguished from S. litura NPV isolates with lowest similarity percentage index (Fig. 4).

 

Biological activity of native SplNPV-FSD15 isolate

Bioassay of isolated strain of SpltNPV against 2nd, 3rd, 4th and 5th instars larvae of S. litura under laboratory condition show a wide range of variation in its biological activity. The LC50 values in three experiments of SpltNPV were inversely correlated with the age of the larvae, LC50 values being the highest for fifth instars. Similarly, LT50 was as low as 69-72 h for second instar larvae and regularly increased to reach at 144 to 146 h for fifth larvae instars. LT50 values were not significantly different between the different independent experiments. Bioassay result revealed that the SpltNPV strain isolated from S. litura was highly effective especially against 2nd, 3rd instar larvae. In first experiment, LC50 values for 2nd , 3rd , 4th and 5th instar larvae were 2.64×103, 2.92×104, 2.94×105, and 2.15×106 OBs/ml, respectively. Briefly, in the third experiment,

 

Table I.- LC50 (95% CL) values of SPLtNPV-FSD15 isolate against 2nd, 3rd and 4th instar of Spodoptera litura.

Instars	SPLtNPV-1					SPLtNPV-2					SPLtNPV-3
	LC50	Slope	X2	df	P	LC50	Slope	X2	df	P	LC50	Slope	X2	df	P
Second	2.64× 103	1029 8210	3.02	1	0.082	1.94× 103	1067 2645	3.07	1	0.080	1.92× 103	1067 2645	3.05	1	0.08
Third	2.92× 104	9727 687	3.87	1	0.049	1.66× 104	1030 1158	4.45	1	0.874	1.91× 104	989 8068	5.46	1	0.01
Fourth	2.94× 105	2481 57064	0.17	1	0.677	2.68× 105	6937 1413	0.09	1	0.755	3.9× 105	5594 7328	0.09	1	0.76
Fifth	2.15× 106	6559 3222	0.44	1	0.503	2.34× 106	5688 8296	0.29	1	0.587	3.96× 106	5457 7160	0.29	1	0.58

Table II.- LT50 (95% CL) value of SPLtNPV-FSD15 isolate against 2nd, 3rd, 4th and 5th instar of Spodoptera litura.

Instars	SPLtNPV-1					SPLtNPV-2					SPLtNPV-3
	LT50 (h)	Slope	X2	df	P	LT50 (h)	Slope	X2	df	P	LT50 (h)	Slope	X2	df	P
Second	72.50	0.91	0.30	1	0.58	70.4	0.94	0.58	1	0.443	69.30	0.95	0.65	1	0.417
Third	95.76	0.80	0.11	1	0.73	98.4	0.78	1.49	1	0.222	93.6	0.83	1.10	1	0.294
Fourth	121.6	0.71	0.00	1	0.95	125.2	0.70	0.00	1	0.950	123.5	0.69	0.11	1	0.731
Fifth	144.6	0.65	0.00	1	0.95	146.8	0.66	0.06	1	0.801	145.8	0.67	0.00	1	0.981

 

for 2nd instars, the most virulent LC50 value (1.92 x 103 POB mL-1) was observed, followed by 2nd and 1st experiment (Table I). The order of LC50 value in the case of three experiments of SpltNPV was second<third <forth <fifth instar. Similarly, LT50 value of 3rd spltNPV experiment was the lowest (59 h) followed by 2nd and 1st experiment (Table II).

 

Discussion

The S. litura (Lepidoptera: Noctuidae) also called armyworm is one of the most destructive and notorious phytophagous insect pests on cotton, vegetables and other field crops. Recently various entomopathogens such as nematodes are being evaluated and utilized to control to S. litura (Safdar et al., 2018). Microbial bio-pesticides based on native baculoviruses have great potential in agriculture with better insecticidal characteristics and higher safety’ for environment. The NPV virus isolates was obtained from diseased S. litura collected cotton. The diseased larvae exhibited viral-like symptoms such as swollen bodies, moribund appearance when field collected and also when laboratory NPV treated. Similarly, because of deposition of occluded viruses (OVs), infected larval bodies become pale and their cuticle ruptured discharging body fluid and ultimately killing the insect. Similar results were reported by Toprak et al. (2005). The viral occlusion bodies (VOBs) of the Pakistani nucleopolyhedrovirus isolates (SlNPV-FSD15) were detected using a light microscope by staining thin smear of infected larvae and drop of VOBs with Giemsa stain which appeared polyhedral and negatively stained particles. The developed bio informative tools and application of latest genomic data facilitated the identification of viruses because of good primer designing. Here, we also used molecular techniques to characterize native S. litura associated NPVs that were found identical to SpltNPVs from other countries and clearly different from Helicoverpa armigera and Mythimna separata NPVs (Kouassai et al., 2009). Further, LC50 values against 2nd -5th instars with LT50 values of this NPV isolate (SltNPV-FSD15) indicated a significant effective control against S. litura. Result obtained by Laarif et al. (2011) indicated that amino acid and nucleotide sequences of S. littoralis (Tun-SlNPV) nucleopolyhedrovirus strain was almost identical with different GenBank deposited sequences of NPVs (Clem et al., 2013; Ikeda et al., 2013).

In bioassay, increasing value of LC50 and LT50 with the age of the host showed the susceptibility of the different larval stages; decline in S. litura susceptibility to NPV is due to dilution effect because larval weight increased as the insect grows (Briese, 1986). Lucein et al. (2009) and Bhutia et al. (2012) also found that LC50 and LT50 values were increased as larval age increased showing - against SpltNPVN. However, Trang et al. (2002) reported that older larvae of S .litura were not affected through ingestion, but intra haemocoelic infection of NPV in mature insect resulted in insect death (Rao et al., 2015). SpltNPV isolate from Biocontrol Research Laboratory (BCRL) was found to be virulent against 3rd instar larvae of S litura larvae with LT50 value (122.16 d) at 1×106 OB’s/ ml (Bhutia et al., 2012). Subramanien et al. (2005) described that LT50 values for larvae of S. litura was dose-dependent. The LC50 value for the larvae of S. litura increases 15,000 times in 2 day-old larvae as compared to 8th day larvae (Trang et al., 2002). Similar trend for LC50 of SpltNPV isolate against 2nd and 3rd were (3.5 9 ×104 and 2.4 9× 105 OBs/ml) observed (Kumar et al., 2011). In summary, increasing dose of NPV caused significant reduction in the survival times of Lepidoptera larvae. This pattern was also observed in instar of Mythima separata and third instars of S. litura larvae (Koussoi et al., 2009). Minimum lethal time (LT50) (96-216 h) of different instars of S. litura suggests that application of SpltNPV isolate as bio insecticide would be economic and attractive strategy for the control of S. litura (Lucein et al., 2009). Contradictory results were found by Koussoi et al. (2009) who observed that high LC50 of MSNPV (180 h) and Indian NPV isolates (240 h) against M. separata made clear that their sole application would not be an efficient strategy for insect control, but should become effective bio insecticides when combined with enhancers. In our case, the very good results of LC50 and LT50 showed highly effectiveness of Slt NPV-FSD15 isolate against all tested instars of S. litura as compared to other reported SltNPV isolates. The biological activity of this isolate is under progress against other important lepidopterous pest of major agriculture crops.

 

Acknowledgements

AMB, JNA, SJNA and IA acknowledge the financial support from PAK 3004 Framework for Pak-Norway Institutional Co-operation Programme (FICP). The authors acknowledge the efforts of Abdullah and late Hafeez ul Rehman for insect collection and rearing of insect culture for experiments.

 

Statement of conflict of interest

The authors have no conflict of interest.

 

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