Maintenance of host insect
Mulberry leaves kept in plastic containers with nylon mesh served as feed for field-collected 1st instar larvae of S. obliqua. The larvae were reared till the adult stage. The eggs laid by the moths were used for maintaining a culture of tested insect to carry out the laboratory experiments. Further, the identity of the pest was confirmed through amplification of cytochrome oxidase I gene (COI) marker (Gupta et al. 2016). The nucleotide sequence was submitted to NCBI GenBank data base (accession number MK531549).
Isolation and mass production of virus
Diseased larvae of S. obliqua, hanging on top branches of jute plants with characteristic viral infection symptoms (Fig. 1), were collected during an epizootic in October 2016 at the research farm of ICAR-Central Research Institute for Jute and Allied Fibers Research, Barrackpore, West Bengal (22.75° N, 88.42°), India. The diseased larvae were brought to ICAR-NBAIR, Bangalore, and were homogenized by distilled water for 3 min, and the crude homogenate was filtered twice by musclin and then the larval debris were removed. The filtrate was spun for 1 min at 500 rpm to remove large particles. The supernatant was re-suspended for 20 min at 5000 rpm and the pellet was collected. Sterile distilled water (100 ml) was used to re-suspend the pellet. The number of occlusion bodies (OBs) in the stock suspension was determined and attained to 3.2 × 104 OBs/ml using a Neubauer hemocytometer.
In vivo mass production of SpobNPV NBAIR1 was carried out on the 5th instar larvae of S. obliqua using plastic trays. About 150 5th instar larvae of S. obliqua were reared on the castor leaves. Castor leaves were washed thoroughly by distilled water, air dried, and smeared with SpobNPV NBAIR1 at 3.2 × 104 OBs/ml using the polished blunt end of a glass rod. Bouquets of these virus-contaminated leaves were placed in plastic trays with a size of 4 cm H × 40 cm L × 30 cm B. Fifth instar larvae of S. obliqua were allowed to feed on these leaves. Once the treated leaves were fully consumed by the larvae, fresh castor leaves were provided. All the dead, virosed larvae were collected and processed after 6-8 days of inoculation. The OBs were quantified by phase-contrast microscopy using a double ruled-improved Neubauer hemocytometer, and the virus suspension was standardized and stored in the refrigerator.
Mortality bioassays
An improved Neubauer hemocytometer was used to estimate the number of occlusion bodies (OBs) in the SpobNPV viral preparation. From the primary stock (5 × 104 OBs/ml), 5 appropriate concentrations were prepared in 0.05% Triton X-100. The surface-sterilized castor leaf discs (5 mm dia) were dipped for 5-10 s in the respective viral dilutions and then air dried vertically. The treated leaf disks were placed in Petri dishes. The final concentration of OBs were estimated at 39.2, 7.8, 1.57, 0.31, and 0.06 OBs/mm2 leaf surface area. Five larvae were released per leaf disc, and 6 replications were maintained for each concentration. The virulence of SpobNPV was tested against early larval instars in independent experiments. Mortality rate was counted 6 days post treatment. The assay was repeated thrice on alternate days. The pooled larval mortality data was subjected to probit analysis using the software POLO (Leora 1994), and the lethal concentration to kill 50% of the test larvae (LC50) was calculated for each larval instar.
Morphological characterization of SpobNPV
Extracted occlusion bodies (OBs) were characterized morphologically by the use of transmission (TECNAI 120 kV TEM (FEI, Netherlands) and scanning (SEM Quanta 250, FEI, Netherlands) electron microscopes. Before visualization, the OBs were processed with fixatives. For primary fixation, suspensions of the occlusion bodies were fixed overnight at 4 °C in vials by using glutaraldehyde 2.5% (v/v) and paraformaldehyde 2% in phosphate buffer (0.1 M, pH 7.4). Then the OBs were washed three times using phosphate buffer 0.1 M (pH 7.4) at 4 °C with a 30 min gap for each wash. Post fixation was carried out for 1 h in aqueous osmium tetroxide (OsO4) 2% (v/v) prepared in 0.1 M phosphate buffer (pH 7.4); washing was done as described for primary fixation. The samples were then dehydrated using ethanol as per Martins et al. (2005) at room temperature with graded ascending series. Critical drying point was achieved by employing dry acetone. Dried samples were placed in stubs and sputter-coated with gold for 20 s in an automated sputter coater (Model: EMITEC - SC 7620). The structure of OBs was viewed in a scanning electron microscope (Quanta 250, FEI, Netherlands) at 3.5 spot size (10 kV and 60 Pa) and captured at ×25,000 magnification and analyzed for actual sizes/shapes of the OBs.
For transmission electron microscope analysis of OBs, the primary fixation of pelleted OBs was done for 24 h at 4 °C in phosphate buffer (0.05 M; pH 7.2) containing 2.5% (v/v) glutaraldehyde. Post fixing was done for 2 h in the same buffer containing 0.5% (v/v) aqueous osmium tetroxide. Washing was done as described under SEM. Dehydrated samples were obtained by using a series of ascending graded alcohol. Three hundred mesh carbon-coated copper grids were used to mount the dehydrated samples. The grids were first stained with uranyl acetate (saturated in water) followed by lead citrate. The grids were observed in a TECNAI 120 kV TEM (FEI, Netherlands). The OBs and nucleocapsids were analyzed morphologically from the magnified electron micrograph by the use of a precision ruler. The shapes and sizes of OBs were determined accordingly.
Molecular characterization of SpobNPV
Purified occlusion bodies (OBs) were obtained by first centrifuging for 1 min at 2500 rpm. The pellet was discarded and supernatant retained as it contained the virus. The supernatant was centrifuged again to allow the OBs to settle on the side walls of centrifuge vial, the OBs thus obtained was dispersed in double-distilled water and kept at −20 °C. For DNA extraction the purified OBs suspension was treated with 1% SDS and proteinase K at 20 mg/ml followed by phenol: chloroform: isoamylalcohol (1:1:1), extraction and DNA was precipitated with ethanol. DNA was re-suspended in TE buffer and extracted DNA was visualized in 0.9% agarose gel. Based on SpobNPV sequences submitted to GenBank, primers were designed for the amplification of species-specific polh gene and the primer sequence consisted of forward primer 5′-ATGCCAGACTTCTCGTACCG-3′ and reverse primer 5′-TAATACGCGGGACCGGTGAAT-3′. The PCR reaction mixture (50 μL) consisted 50 ng of template DNA, 0.25 U of Taq DNA polymerase, 10X Taq buffer, 2.5 mM MgCl2, 2.5 mM of each of four dNTPs, and 1 μL each forward and reverse primer. The reaction was carried out in a thermal cycler (Biorad, Model T100) with initial denaturation at 95 °C for 3 min and 35 cycles at 95 °C for 30 sec, 70 °C for 1 min, 55 °C for 1 min, and a final extension step at 72 °C for 10 min to obtain a PCR product of 700 bp. The PCR products were sequenced and submitted to NCBI GenBank.
Host range studies
The host range of the SpobNPV NBAIR1 was studied on a 1 day old 3rd larvae of S. litura, S. exigua, S. frugiperda, Helicoverpa armigera, Plutella xylostella, Amsacta albistriga, Maruca vitrata, Trichoplusia ni, Pieris brassicae, Agrotis ipsilon, and Bombyx mori with SpobNPV NBAIR1 at 3.2 × 104 OBs/ml concentration. The bioassays were scored 6 days post treatment to access the viral infection. All the dead larvae in the course of the assays were examined for the presence of virus OBs by smearing the larval body fluid on a glass slide, stained with Giemsa stain, and examined using a phase-contrast light microscope under oil immersion.
Field testing of SpobNPV NBAIR1
Two field experiments were conducted at ICAR-Central Research Institute for Jute and Allied Fibers Research farm, Barrackpore, Kolkata, India, during the years 2017 and 2018, on jute to evaluate the efficacy of the formulation of SpobNPV strain NBAIR1 during the peak time of S. obliqua infestation. The field experiment was laid out in a randomized block design with a plot size of 3 × 4 m and with 4 replications. Field efficacy of SpobNPV was compared to Bacillus thuringiensis var. kurstaki (Btk) NCIM2514 at 2 ml/l (Lipel®), Agrilife Private Limited, Hyderabad, India, and conventional insecticide profenophos 50% EC at 2 ml/l. Jute seeds (variety JRO524) were sown during mid monsoon season (mid-July) with a row-row space of 15 cm and plant to plant space of 30 cm apart after thinning. The crop was raised following the recommended agronomical practices. Pre-treatment observation was recorded at 1 day before spraying on the number of larvae from 25 randomly selected plants. Treatments were imposed to plants with knapsack sprayer when early instar larvae were noticed. SpobNPV at 1.5 × 1011 OBs/ha with jaggery 5%, glycerol 10%, and Sandovit 0.1 % was sprayed at 2 ml/l during evening hours. Btk NCIM2514 was sprayed at 2 ml/l. Post treatment observations on the number of S. obliqua larvae were recorded at 3, 4, and 7 days after the SpobNPV application from 25 randomly selected plants.
Statistical analysis
ANOVA was used as a statistical tool for Agricultural Research (STAR, Version 2.0.1, developed by International Rice Research Institute, Manila, Philippines, 2013 to 2020. This software is used for statistical analysis.